Team:KIT-Kyoto/Notebook/LabNote/GFPMLF9

From 2011.igem.org

(Difference between revisions)

Latest revision as of 03:45, 6 October 2011

Home	Team	Project	Parts	Notebook	Safety	Human Practice	Attributions

Home > Notebook > Lab Note > September	Language：English/Japanese

1st, September

Member

Nakagawa

1.Using [http://www.qiagen.com/products/dnacleanup/gelpcrsicleanupsystems/qiaquickgelextractionkit.aspx|QIAquick Gel Extraction Kit] and extracted DNA of pSB1C3 from the gel.

2.DNA bands in the agarose gel.

After extracting DNA,I measured 5µl ,and diluted it for 20 times.

And I measured its density.

3.PCR reaction was carried out by using primers designed on August 30th. The reaction conditions are summarized below.

PCR reaction

10 µM Primer F	1.5 µl
10 µM Primer R	1.5 µl
BBa_E0240	1 µl
10 x PCR Buffer for KOD Plus	5 µl
dNTPs	4 µl
MgSO₄	4 µl
ddH₂O	32 µl
KOD Plus	1 µl
	total 50 µl

Cycle

Pre-Denature	95°C	30sec
Denature	95°C	30sec	30 Cycle
Anneling	48.5°C	1min
Extension	68°C	1kb/min
End	4°C	keep

Results

2.Image of the agarose gel.

You can see DNA band at around 2kbp.

density(ng/µl)

1	19.0
2	13.0
3	19.0
4	22.0
5	16.0
ave.	17.8

2nd, September

Member

Nakagawa

Isolate BBa_E0240 by phenol-chloroform treatment,Agarose gel electrophoresis and carried out to detect the PCR products.

I digested PCR products with the following restriction enzymes (at 37°C for 16 hours).

ddH₂O	2 µl
BBa_E0240	50 µl
EcoRⅠ	1 µl
PstⅠ	1 µl
10 x H Buffer	6 µl
	total 60 µl

Results

2.Image of the agarose gel.

I can see BBa_E0240 band at the correct place.And the density was enough to watch it.

3rd,September

Member

Nakagawa

Using [http://www.qiagen.com/products/dnacleanup/gelpcrsicleanupsystems/qiaquickgelextractionkit.aspx|QIAquick Gel Extraction Kit] and extracted DNA of pSB1C3 from the gel.

Results

5th,September

Member

Nakagawa

Ligate BBa_E0240.which doesn’t have ATG codons and pSB1C3

I have transformed E. coli DH5 alpha with it.

Before I made BBa_E0240.which doesn’t have ATG codons and pSB1C3.

The densities are 19ng/µl and 25ng/µl.

DNA samples were digested by the following restriction enzymes at 37 ℃ for 30 min.

BBa_E0240	0.5 µl
pSB1C3	0.5 µl
10 x Buffer	2.5 µl
F4 ligase	0.5 µl
ddH₂O	1.0 µl
	total 5 µl

After ligate them, I have transformed E. coli DH5 alpha with it.

Results

There aren’t any colonies on the plate.

However I heared the success probability is very low, so next time I want to be careful about the density.

6th,September

Member

Yoshimura,Nakagawa

1.Ligate BBa_E0240.which doesn’t have ATG codons and pSB1C3

DNA samples were digested by the following restriction enzymes at 37 ℃ for 30 min

insert	0.5 µl
vector	0.5 µl
10 x Buffer	2.5 µl
F4 ligase	0.5 µl
ddH₂O	1.0 µl
	total 5 µl

However, I ligated them with this density ratio (bector: insert=1:9)

After ligate them, I have transformed E. coli DH5 alpha with it.

2. We attached restriction enzyme site of EcoRⅠand XbaⅠto the Flag-tag dMLF from pUAST Flag-tag dMLF.

We isolated Flag-tag dMLF from pUAST Flag-tag dMLF.

Results

1.There are 4 colonies on the LB plate.

2.F：AAAGAATTCAAATCTAGAAAAATGGACTACAAGGACGA

　　　　　 EcoRⅠ　　XbaⅠ

Tm value:72.14℃ 38bases

R：AAACTGCAGAAAACTAGTAAATACCCTACTTCTTCTTGCC

　　　　 PstⅠ　　　SpeⅠ

Tm value:72.16℃　40bases

7th.September

Member

Nakagawa

I did pre-culture of yesterday colonies and pSB1C3.

PCR and restriction enzyme with MLF

PCR reaction

10 µM GFP Primer F	1.5 µl
10 µM GFP Primer R	1.5 µl
BBa_E0240	1 µl
10 x PCR Buffer for KOD Plus	5 µl
dNTPs	4 µl
MgSO₄	4 µl
ddH₂O	32 µl
KOD Plus	1 µl
	total 50 µl

Cycle

Pre-Denature	95°C	30sec
Denature	95°C	30sec	30 Cycle
Anneling	50°C	1min
Extension	68°C	1min
End	4°C	keep

After that, I isolated MLF with restriction enzyme.

DNA samples were digested by the following restriction enzymes at 37 ℃ for 16 hours.

ddH₂O	2 µl
Flag tag dMLF	50 µl
EcoRⅠ	1 µl
PstⅠ	1 µl
10 x H Buffer	6 µl
	total 60 µl

8th,September

Member

Nakagawa

Using [http://www.qiagen.com/products/dnacleanup/gelpcrsicleanupsystems/qiaquickgelextractionkit.aspx|QIAquick Gel Extraction Kit] and extracted DNA of Flag tag dMLF from the gel.

Isolatioe pSB1C3 by the alkaline-lysis method by phenol-chloroform treatment, DNA was digested with the following restriction enzymes (30 min at 37℃).

I digested the DNA of pSB1C3 with the following restriction enzymes (at 37°C for 16 hours).

ddH₂O	2 µl
pSB1C3	50 µl
EcoRⅠ	1 µl
PstⅠ	1 µl
10 x H Buffer	6 µl
	total 60 µl

Results

We cannot see any bands of MLF from the gel.

So, we are effort to ligate with pSB1C3 and BBa_E0240.

9th,September

Member

Nakagawa

I planned to give efficiency of the ligation by giving rise to the density of GFP and vector by ethanol precipitating

Results

I failed to rise the densities of vector and the DNA of insert.

We lost DNA In the middle of ethanol precipitation.

12th,September

Members

Yoshimura, Nakagawa

1.We amplified the Flag-MLF by using primer which was made on September 6th.

We did PCR reaction with following conditions.

PCR reaction(1)

10 µM Primer F	1.5 µl
10 µM Primer R	1.5 µl
Template DNA	1 µl
10 x PCR Buffer for KOD Plus	5 µl
dNTPs	4 µl
MgSO₄	4 µl
ddH₂O	32 µl
KOD Plus	1 µl
	total 50 µl

Cycle

Pre-Denature	94°C	2min
Denature	94°C	15sec	35 Cycle
Anneling	55°C	30sec
Extension	68°C	1min 20sec
End	4°C	keep

PCR reaction(2)

10 µM Primer F	1.5 µl
10 µM Primer R	1.5 µl
Template DNA	1 µl
10 x PCR Buffer for KOD Plus	5 µl
dNTPs	4 µl
MgSO₄	2 µl
ddH₂O	34 µl
KOD Plus	1 µl
	total 50 µl

Cycle

Pre-Denature	94°C	2min
Denature	94°C	15sec	35 Cycle
Anneling	55°C	30sec
Extension	68°C	1min 20sec
End	4°C	keep

We collected each sample and kept them in -20°C.

2.Pre-culture of E.coli(DH5α)which contains pSB1C3.

I tried doing PCR reaction with following conditions with BBa_E0240 again.

PCR reaction

10 µM Primer F	1.5 µl
10 µM Primer R	1.5 µl
GFP	1 µl
10 x PCR Buffer for KOD Plus	5 µl
dNTPs	4 µl
MgSO₄	4 µl
ddH₂O	32 µl
KOD Plus	1 µl
	total 50 µl

Cycle

Pre-Denature	95°C	30sec
Denature	95°C	30sec	30 Cycle
Anneling	48.5°C	1min
Extension	68°C	1kb/min
End	4°C	keep

I isolated pSB1C3 by phenol-chloroform treatment .I digested DNA with the following restriction enzymes (30 minutes at 37°C).

ddH₂O	2 µl
BBa_E0240	50 µl
EcoRⅠ	1 µl
PstⅠ	1 µl
10 x H Buffer	6 µl
	total 60 µl

Results

The E.coli (DH5α) which contains pSB1C3 was increased, but there are some colonies which doesn’t correct color.

Possibly deterioration may begin in plate in itself.

The DNA of PCR reaction increased next day.

13th,September

Member

Yoshimura, Nakagawa

1.We carried out agarose gel electrophoresis to detect the PCR products.

2.We amplified Flag-tag dMLF by using the primer which I manufactured on 6th September.

Restriction enzyme processing.

This is because after examining a sequence of Flag-tag dMLF, a restriction enzyme site of PstI and XbaI was seen.

So, I examined whether PCR product of Flag-tag dMLF is cut by PstI and XbaI.

(1)

MilliQ	6.5 µl
Flag-tag dMLF	20 µl
PstⅠ	0.5 µl
10 x H Buffer	3 µl
	total 30 µl

(2)

MilliQ	6.5 µl
Flag-tag dMLF	20 µl
XbaⅠ	0.5 µl
10 x M Buffer	3 µl
	total 30 µl

(3)

MilliQ	6 µl
Flag-tag dMLF	20 µl
PstⅠ	0.5 µl
XbaⅠ	0.5 µl
10 x M Buffer	3 µl
	total 30 µl

After incubate it in 37°C for 50 minutes, I performed electrophoresis with agarose gel.

Photograph after the electrophoresis.

3.To amplify Flag-tag dMLF cDNA, PCR reactions were carried out under the following conditions.

PCR reaction

10 µM Primer F	1.5 µl
10 µM Primer R	1.5 µl
Template DNA	1 µl
10 x PCR Buffer for KOD Plus	5 µl
dNTPs	4 µl
MgSO₄	4 µl
ddH₂O	32 µl
KOD Plus	1 µl
	total 50 µl

Cycle

Pre-Denature	94°C	2min
Denature	94°C	15sec	35 Cycle
Anneling	55°C	30sec
Extension	68°C	1min 20sec
End	4°C	keep

We collected each sample and kept them in -20°C.

Results

1.Image of the agarose gel.

2.Images of the agarose gel.

:

Flag-tag dMLF aren’t cut by restriction enzyme of PstI and XbaI.

A marker did not enough to drift.

14th,September

Members

Yoshimura

1.We carried out agarose gel electrophoresis to detect the PCR products.

2.I extracted GFP, DNA of the vector from gel using [http://www.qiagen.com/products/dnacleanup/gelpcrsicleanupsystems/qiaquickgelextractionkit.aspx|QIAquick Gel Extraction Kit].

Results 1.Image of the agarose gel.

After doubling the density of gel, a marker did not drift.

I will decide to perform it with 2% of agarose gel about the electrophoresis of Flag-tag MLF in future.

2.I measured the density with an absorbance meter afterwards.

GFP

1	0.158
2	0.147
3	0.160
4	0.159
5	0.158
ave.	0.156

pSB1C3

1	0.029
2	0.021
3	0.028
4	0.022
5	0.025
ave.	0.025

GFP

pSB1C3

The BBa_E0240 succeeded in the extraction from gel and recorded 156ng/µl by the measurement with the absorbance meter and the pSB1C3 became 25ng/µl.

Therefore I decided to begin an experiment of the ligation again from the next day.

17th,September

Members

Nakagawa

1.Ligate with BBa_E0240 and pSB1C3

2.Pre-culture and the alkaline-lysis method of pSB1C3

I adjusted reaction liquid according to the following composition.

pSB1C3:GFP＝1:2

insert	0.3 µl
vector	2.0µl
10 x Buffer	2.5 µl
F4 ligase	0.5 µl
ddH₂O	1.7 µl
	total 7 µl

pSB1C3:GFP＝1:5

insert	1.0 µl
vector	1.0 µl
10 x Buffer	2.5 µl
F4 ligase	0.5 µl
ddH₂O	2.0 µl
	total 7 µl

pSB1C3:GFP＝1:10

insert	1.3 µl
vector	1.0 µl
10 x Buffer	2.5 µl
F4 ligase	0.5 µl
ddH₂O	1.7 µl
	total 7 µl

I incubated them in 16°C for 30 min.

After that, I transformed them, and I poured them into 300 µl of SOC medium and add them on the plate after culture for 30 minutes.

I thought that I tried to pick at only a red colony as a point to keep in mind of this time and lowered the possibility of contaminating it.

Results

Here are many colonies on the LB medium.

Possibly contaminating is thought for example an antibiotic does not work.

The pSB1C3 was not able to be refined again.

19th,September

Members

Yoshimura Nakagawa

1.We transformed and performed restriction enzyme processing

2.Density check of BBa_E0240 and pSB1C3

3.Pre-culture with yesterday colony and pSB1C3 to the new LB plate

I digested with the XhoI according to the following composition.

(1)

MilliQ	6.5 µl
Flag-tag dMLF	20 µl
PstⅠ	0.5 µl
10 x H Buffer	3 µl
	total 30 µl

(2)

MilliQ	6.5 µl
Flag-tag dMLF	20 µl
XbaⅠ	0.5 µl
10 x M Buffer	3 µl
	total 30 µl

(3)

MilliQ	6 µl
Flag-tag dMLF	20 µl
PstⅠ	0.5 µl
XbaⅠ	0.5 µl
10 x M Buffer	3 µl
	total 30 µl

After incubate it in 37 ゜ C for 18 hours, I performed agarose gel electrophoresis.

Checking the amount of the purified GFP,

1 x	1µl
2 x	1µl
4 x	1µl
8 x	1µl

Image of agarose gel after DNA fragment isolation for Checking the amount of the purified GFP.

Results

The increase of BBa_E0240 was good, and upbringing of the big colony was seen.

pSB1C3 was not BBa_E0240 too, but was all right because some number grew.

24th,September

Members

Yoshimura Nakagawa

1.alkaline-lysis method, phenol-chloroform treatment

2.restriction enzymes, extraction with pSB1C3

3.restriction enzymes processing with BBa_E0240

Yield rose markedly when I exchanged isopropanol.

After alkaline-lysis method, phenol-chloroform treatment with pSB1C3,restriction enzymes processing with BBa_E0240 and pSB1C3

According to a list shown below, I performed restriction enzyme processing at 37 overnight.

ddH₂O	2 µl
BBa_E0240	50 µl
EcoRⅠ	1 µl
PstⅠ	1 µl
10 x H Buffer	6 µl
	total 60 µl

The restriction enzyme-digested pSB1C3 were extracted from the agarose gel by QIAquick Gel Extraction Kit.

Photograph after the extraction.

Results

Image of agarose gel after DNA fragment isolation.

A band was seen to about 2kb.

26th,September

Members

Yoshimura Nakagawa

1.Ligation

2.restriction enzyme handling of liquid pSB1C3

restriction enzymes processing with pSB1C3

According to a list shown below, I performed restriction enzyme processing at 37 for 30 minutes.

ddH₂O	2 µl
BBa_E0240	50 µl
EcoRⅠ	1 µl
PstⅠ	1 µl
10 x H Buffer	6 µl
	total 60 µl

Next ligate with following list.

At first I dilute the density of vector to become 10 pg/µl.

And I dilute it as I calculate one and MLF 60 ng/µl of GFP 70 ng/µl to be able to go by combination below and am composed follows at 1 µl.

I combined it in the ratio of follows afterwards.

pSB1C3:GFP＝1:10

insert	1.0 µl
vector	1.0µl
10 x Buffer	2.5 µl
F4 ligase	0.5 µl
ddH₂O	1.0 µl
	total 6.0 µl

pSB1C3:GFP＝1:20

insert	1.0 µl
vector	1.0 µl
10 x Buffer	2.5 µl
F4 ligase	0.5 µl
ddH₂O	1.0 µl
	total6.0µl

pSB1C3:GFP＝1:40

insert	1.0 µl
vector	1.0 µl
10 x Buffer	2.5 µl
F4 ligase	0.5 µl
ddH₂O	1.0 µl
	total 6 µl

I incubated them in 16°C for 30 min.

After that, I transformed them, and I poured them into 300 µl of SOC medium and add them on the plate after culture for 30 minutes.

Results

There are no colonies.

29th,September

Members

Yoshimura, Nakagawa

At first I dilute the density of vector to become 10 pg/µl.

And I dilute it as I calculate one and MLF 60 ng/µl of GFP 70 ng/µl to be able to go by combination below and am composed follows at 1 µl.

I combined it in the ratio of follows afterwards.

pSB1C3:GFP＝1:10

insert	1.0 µl
vector	1.0µl
10 x Buffer	2.5 µl
F4 ligase	0.5 µl
ddH₂O	1.0 µl
	total 6.0 µl

pSB1C3:GFP＝1:20

insert	1.0 µl
vector	1.0 µl
10 x Buffer	2.5 µl
F4 ligase	0.5 µl
ddH₂O	1.0 µl
	total6.0µl

pSB1C3:GFP＝1:40

insert	1.0 µl
vector	1.0 µl
10 x Buffer	2.5 µl
F4 ligase	0.5 µl
ddH₂O	1.0 µl
	total 6 µl

I incubated them in 16°C for 30 min.

After that, I transformed them, and I poured them into 300 µl of SOC medium and add them on the plate after culture for 30 minutes.

Results

There are some colonies on the plate.

However there aren’t any MLF colonies.

30th,September

Members

Yoshimura, Nakagawa

At first I dilute the density of vector to become 10 pg/µl

And I dilute it as I calculate one and MLF 60 ng/µl of GFP 70 ng/µl to be able to go by combination below and am composed follows at 1 µl

I combined it in the ratio of follows afterwards

pSB1C3:GFP＝1:10

insert	1.0 µl
vector	1.0µl
10 x Buffer	2.5 µl
F4 ligase	0.5 µl
ddH₂O	1.0 µl
	total 6.0 µl

pSB1C3:GFP＝1:20

insert	1.0 µl
vector	1.0 µl
10 x Buffer	2.5 µl
F4 ligase	0.5 µl
ddH₂O	1.0 µl
	total6.0µl

pSB1C3:GFP＝1:40

insert	1.0 µl
vector	1.0 µl
10 x Buffer	2.5 µl
F4 ligase	0.5 µl
ddH₂O	1.0 µl
	total 6 µl

I incubated them in 16°C for 30 min.

After that, I transformed them, and I poured them into 300 µl of SOC medium and add them on the plate after culture for 30 minutes.

After that We did alkaline-lysis method restriction and enzymes processing with ligation products.

Results

After all MLF did not exist even if there was the BBa_E0240 in ligation products.

And the first turn of parts was completed today.

@@ Line 19: / Line 19: @@
 : After extracting DNA,I measured 5µl ,and diluted it for 20 times.
 : And I measured its density.
-:3.PCR reaction was carried out by using primers designed on August 30rd. The reaction conditions are summarized below.
+:3.PCR reaction was carried out by using primers designed on August 30th. The reaction conditions are summarized below.
 :<table border="0"><tr><td>
@@ Line 33: / Line 33: @@
 :<tr><td align=center>MgSO<sub>4</sub></td><td align=right>4 µl</td></tr>
 :<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>32 µl</td></tr>
-:<t><td align=center>KOD Plus</td><td align=right>1 µl</td></tr>
+:<tr><td align=center>KOD Plus</td><td align=right>1 µl</td></tr>
 :<tr><td>&nbsp;</td><td align=right>total 50 µl</td></tr>
 :</table>
@@ Line 58: / Line 58: @@
 :You can see DNA band at around 2kbp.
 <br>
-:density
+:density(ng/µl)
 :<table border=1 width="160px">
-:<tr><td width="70px" align=center>1</td><td width="90px"  align=right>0.019</td></tr>
+:<tr><td width="70px" align=center>1</td><td width="90px"  align=right>19.0</td></tr>
-:<tr><td align=center>2</td><td align=right>0.013</td></tr>
+:<tr><td align=center>2</td><td align=right>13.0</td></tr>
-:<tr><td align=center>3</td><td align=right>0.019</td></tr>
+:<tr><td align=center>3</td><td align=right>19.0</td></tr>
-:<tr><td align=center>4</td><td align=right>0.022</td></tr>
+:<tr><td align=center>4</td><td align=right>22.0</td></tr>
-:<tr><td align=center>5</td><td align=right>0.016</td></tr>
+:<tr><td align=center>5</td><td align=right>16.0</td></tr>
-:<tr><td align=center>ave.</td><td align=right>0.0178</td></tr>
+:<tr><td align=center>ave.</td><td align=right>17.8</td></tr>
 :</table>
-<br>
 ==''2nd, September''==
@@ Line 94: / Line 93: @@
 <br>
 :I can see BBa_E0240 band at the correct place.And the density was enough to watch it.
-<br>
 ==''3rd,September''==
@@ Line 108: / Line 109: @@
 " width="250px" height="250px" border="0">
 </body></html>
-<br>
 ==''5th,September''==
@@ Line 116: / Line 119: @@
 <br>
 :Ligate BBa_E0240.which doesn’t have ATG codons and pSB1C3
-::I have transformed E. coli DH5 alpha with it.
+::I have transformed ''E. coli DH5 alpha'' with it.
 ::Before I made BBa_E0240.which doesn’t have ATG codons and pSB1C3.
 ::The densities are 19ng/µl and 25ng/µl.
@@ Line 135: / Line 138: @@
 :There aren’t any colonies on the plate.
 :However I heared the success probability is very low, so next time I want to be careful about the density.
-<BR>
-==''6th September''==
+==''6th,September''==
 <b>Member</b>
 <br>
 :Yoshimura,Nakagawa
 <br>
-Ligate BBa_E0240.which doesn’t have ATG codons and pSB1C3
+:1.Ligate BBa_E0240.which doesn’t have ATG codons and pSB1C3
-I have transformed E. coli DH5 alpha with it.
+: DNA samples were digested by the following restriction enzymes at 37 ℃ for 30 min
-We isolated Flag-tag dMLF from pUAST Flag-tag dMLF.
+:<table border=1 width="200px">
+:<tr><td width="130px" align=center>insert</td><td width="70px" align=right>0.5 µl</td></tr>
+:<tr><td align=center>vector</td><td align=right>0.5 µl</td></tr>
+:<tr><td align=center>10 x Buffer</td><td align=right>2.5 µl</td></tr>
+:<tr><td align=center>F4 ligase</td><td align=right>0.5 µl</td></tr>
+:<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>1.0 µl</td></tr>
+:<tr><td>&nbsp;</td><td align=right>total 5 µl</td></tr>
+:</table>
-We will make new parts.
+:However, I ligated them with this density ratio (bector: insert=1:9)
-DNA samples were digested by the following restriction enzymes at 37 ℃ for 30 min
+:After ligate them, I have transformed E. coli DH5 alpha with it.
-表を表示
+:2. We attached restriction enzyme site of EcoRⅠand XbaⅠto the Flag-tag dMLF from pUAST Flag-tag dMLF.
+:We isolated Flag-tag dMLF from pUAST Flag-tag dMLF.
+<br>
+<b>Results</b>
+:1.There are 4 colonies on the LB plate.
+:2.F：AAA<font color="#ff0000">GAATTC</font>AAA<font color="#ff0000">TCTAGA</font>AAAATGGACTACAAGGACGA<br>
+:　　　　　      <em>Eco</em>RⅠ　　<em>Xba</em>Ⅰ<br>
+:Tm value:72.14℃  38bases
-However, I ligated them with this density ratio (bector: insert=1:9)
+:R：AAA<font color="#ff0000">CTGCAG</font>AAA<font color="#ff0000">ACTAGT</font>AAATACCCTACTTCTTCTTGCC<br>
+:　　　　 <em>Pst</em>Ⅰ　　　<em>Spe</em>Ⅰ<br>
+:Tm value:72.16℃　40bases
-After ligate them, I have transformed E. coli DH5 alpha with it.
-And We attached restriction enzyme site of EcoRⅠand XbaⅠto the Flag-tag dMLF from pUAST Flag-tag dMLF.
-F：AAAGAATTCAAATCTAGAAAAATGGACTACAAGGACGA
-EcoRⅠ     XbaⅠ
-Tm value:72.14℃  38bases
-R：AAACTGCAGAAAACTAGTAAATACCCTACTTCTTCTTGCC
-　　　　　PstⅠ　　　　　SpeⅠ
-Tm value:72.16℃　40bases
-Result
+==''7th.September''==
-There are 4 colonies on the LB plate.
+<b>Member</b>
+<br>
+:Nakagawa
+<br>
+:I did pre-culture of yesterday colonies and pSB1C3.
+:PCR and restriction enzyme with MLF
+:<table border="0"><tr><td>
+:<table border="0" width="150px">
+:<tr><td align=center>PCR reaction</td></tr>
+:</table>
+:<table border=1 width="250px">
+:<tr><td width="150px" align=center>10 µM GFP Primer F</td><td width="100px"  align=right>1.5 µl</td></tr>
+:<tr><td align=center>10 µM GFP Primer R</td><td align=right>1.5 µl</td></tr>
+:<tr><td align=center>BBa_E0240</td><td align=right>1 µl</td></tr>
+:<tr><td align=center>10 x PCR Buffer for KOD Plus</td><td align=right>5 µl</td></tr>
+:<tr><td align=center>dNTPs</td><td align=right>4 µl</td></tr>
+:<tr><td align=center>MgSO<sub>4</sub></td><td align=right>4 µl</td></tr>
+:<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>32 µl</td></tr>
+:<tr><td align=center>KOD Plus</td><td align=right>1 µl</td></tr>
+:<tr><td>&nbsp;</td><td align=right>total 50 µl</td></tr>
+:</table>
+:</td><TD></TD><TD></TD><td>
+:<table border="0" width="100px">
+:<tr><td align=center>Cycle</td></tr>
+:</table>
+:<table border=1 width="400px">
+:<tr><td width="100px" align=center>Pre-Denature</td><td width="100px" align=center>95°C</td><td width="100px" align=center>30sec</td><td width="100px">&nbsp;</td></tr>
+:<tr><td align=center>Denature</td><td align=center>95°C</td><td align=center>30sec</td><td align=center rowspan=3>30 Cycle</td></tr>
+:<tr><td align=center>Anneling</td><td align=center>50°C</td><td align=center>1min</td></tr>
+:<tr><td align=center>Extension</td><td align=center>68°C</td><td align=center>1min</td></tr>
+:<tr><td align=center>End</td><td align=center>4°C</td><td align=center>keep</td><td width="100px">&nbsp;</td></tr>
+:</table>
+:</td></tr>
+:</table>
+<BR>
+:After that, I isolated MLF with restriction enzyme.
+:DNA samples were digested by the following restriction enzymes at 37 ℃ for 16 hours.
-<BR>
+:<table border=1 width="220px">
-<BR>
+:<tr><td width="130px" align=center>ddH<sub>2</sub>O</td><td width="90px"  align=right>2 µl</td></tr>
-<BR>
+:<tr><td align=center>Flag tag dMLF</td><td align=right>50 µl</td></tr>
-<BR>
+:<tr><td align=center><em>Eco</em>RⅠ</td><td align=right>1 µl</td></tr>
-<BR>
+:<tr><td align=center><em>Pst</em>Ⅰ</td><td align=right>1 µl</td></tr>
-/6(火)★おｋ★<BR>
+:<tr><td align=center>10 x H Buffer</td><td align=right>6 µl</td></tr>
-吉村、中川<BR>
+:<tr><td align=center>&nbsp;</td><td align=right>total 60 µl</td></tr>
-<BR>
+:</table>
-pSB1C3と開始コドン抜きGFPのライゲーションと形質転換<BR>
-【目的】<BR>
-新しいiGEMパーツの製作<BR>
-<BR>
-【実験方法】<BR>
-°C、30minでincubateする<BR>
+==''8th,September''==
+<b>Member</b>
+<br>
+:Nakagawa
+<br>
+:Using [http://www.qiagen.com/products/dnacleanup/gelpcrsicleanupsystems/qiaquickgelextractionkit.aspx|QIAquick Gel Extraction Kit] and extracted DNA of Flag tag dMLF from the gel.
-下記の組成に従って反応液を調整した<BR>
+:Isolatioe pSB1C3 by the alkaline-lysis method by phenol-chloroform treatment, DNA was digested with the following restriction enzymes (30 min at 37℃).
-<table border=1 width="200px">
+:I digested the DNA of pSB1C3 with the following restriction enzymes (at 37°C for 16 hours).
-<tr><td width="130px" align=center>insert</td><td width="70px" align=right>0.5 µl</td></tr>
-<tr><td align=center>vector</td><td align=right>0.5 µl</td></tr>
-<tr><td align=center>10 x Buffer</td><td align=right>2.5 µl</td></tr>
-<tr><td align=center>F4 ligase</td><td align=right>0.5 µl</td></tr>
-<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>1.0 µl</td></tr>
-<tr><td>&nbsp;</td><td align=right>total 5 µl</td></tr>
-</table>
-<BR>
+:<table border=1 width="220px">
+:<tr><td width="130px" align=center>ddH<sub>2</sub>O</td><td width="90px"  align=right>2 µl</td></tr>
+:<tr><td align=center>pSB1C3</td><td align=right>50 µl</td></tr>
+:<tr><td align=center><em>Eco</em>RⅠ</td><td align=right>1 µl</td></tr>
+:<tr><td align=center><em>Pst</em>Ⅰ</td><td align=right>1 µl</td></tr>
+:<tr><td align=center>10 x H Buffer</td><td align=right>6 µl</td></tr>
+:<tr><td align=center>&nbsp;</td><td align=right>total 60 µl</td></tr>
+:</table>
+<br>
+<b>Results</b>
+:We cannot see any bands of MLF from the gel.
+:So, we are effort to ligate with pSB1C3 and BBa_E0240.
-°C、30minでincubateした<BR>
-今回はベクターとインサートの濃度比を1:9にしてライゲーションを行った。<BR>
-昨日と同様にトラフォを行うことにした<BR>
-<BR>
-トランスフォーメーション<BR>
-<BR>
-<BR>
-【実験結果】<BR>
-つのコロニーが確認できた。<BR>
-<BR>
-<BR>
+==''9th,September''==
+<b>Member</b>
 <br>
-プライマー設計<br>
+:Nakagawa
-【目的】<br>
-pUAST Fiag-tag dMLFからFiag-tag dMLFを単離・増殖させる。<br>
 <br>
-【実験方法】<br>
+:I planned to give efficiency of the ligation by giving rise to the density of GFP and vector by ethanol precipitating
-ｐUAST Fiag-tag dMLF<span style="WIDOWS: 2; TEXT-TRANSFORM: none; BACKGROUND-COLOR: rgb(255,255,255); TEXT-INDENT: 0px; LETTER-SPACING: normal; FONT: 13px/19px sans-serif; WHITE-SPACE: normal; ORPHANS: 2; COLOR: rgb(0,0,0); WORD-SPACING: 0px; -webkit-text-decorations-in-effect: none; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px" class=Apple-style-span>からの Fiag-tag
-dMLFの上流に<em>EcoR</em>Ⅰ、<em>Xba</em>Ⅰの制限酵素サイト、下流に<em>Spe</em>Ⅰ、<em>Pst</em><span class="Apple-converted-space">&nbsp;</span>Ⅰの制限酵素サイトがあるようにプライマー設計をした。<span style="WIDOWS: 2; TEXT-TRANSFORM: none; BACKGROUND-COLOR: rgb(223,223,223); TEXT-INDENT: 0px; LETTER-SPACING: 1px; FONT: 13px/19px Tahoma; WHITE-SPACE: normal; ORPHANS: 2; COLOR: rgb(0,0,0); WORD-SPACING: 0px; -webkit-text-decorations-in-effect: none; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px" class=Apple-style-span><br>
-</span></span><br>
 <br>
-【実験結果】<br>
+<b>Results</b>
-F：AAA<font color="#ff0000">GAATTC</font>AAA<font color="#ff0000">TCTAGA</font>AAAATGGACTACAAGGACGA<br>
+:I failed to rise the densities of vector and the DNA of insert.
-　　　　　<em>Eco</em>RⅠ　　　　<em>Xba</em>Ⅰ<br>
+:We lost DNA In the middle of ethanol precipitation.
-Tm値:72.14℃　38塩基<br>
-</span></p>
-<p><span style="WIDOWS: 2; TEXT-TRANSFORM: none; BACKGROUND-COLOR: rgb(255,255,255); TEXT-INDENT: 0px; LETTER-SPACING: normal; FONT: 13px/19px sans-serif; WHITE-SPACE: normal; ORPHANS: 2; COLOR: rgb(0,0,0); WORD-SPACING: 0px; -webkit-text-decorations-in-effect: none; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px" class=Apple-style-span>R：AAA<font color="#ff0000">CTGCAG</font>AAA<font color="#ff0000">ACTAGT</font>AAATACCCTACTTCTTCTTGCC<br>
-　　　　　<em>Pst</em>Ⅰ　　　　　<em>Spe</em>Ⅰ<br>
-Tm値:72.16℃　40塩基<br>
-<br>
-<br>
-<BR>
-<BR>
-<BR>
-/7(水)★おｋ★<BR>
-中川<BR>
-(目的)<BR>
-・昨日ライゲーションしてできたコロニーのプレカルチャーをした。<BR>
-・MLFのPCRと制限酵素処理<BR>
-・pSB1C3のプレカルチャー<BR>
-(方法)<BR>
-昨日のライゲーションで4つのコロニーができていたのでプレカルチャーを行うことにした<BR>
-<BR>
-<BR>
-<BR>
-それと同時進行で白血病の原因タンパクを作るとされているMLFをPCRで増やすことを行った
-PCR(MLF)<BR>
-<table border="0"><tr><td>
-<table border="0" width="150px">
-<tr><td align=center>PCR条件</td></tr>
-</table>
-<table border=1 width="220px">
-<tr><td width="150px" align=center>10 µM Primer F</td><td width="70px"  align=right>1.5 µl</td></tr>
-<tr><td align=center>10 µM Primer R</td><td align=right>1.5 µl</td></tr>
-<tr><td align=center>MLF</td><td align=right>1 µl</td></tr>
-<tr><td align=center>10 x PCR Buffer for KOD Plus</td><td align=right>5 µl</td></tr>
-<tr><td align=center>dNTPs</td><td align=right>4 µl</td></tr>
-<tr><td align=center>MgSO<sub>4</sub></td><td align=right>4 µl</td></tr>
-<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>32 µl</td></tr>
-<tr><td align=center>KOD Plus</td><td align=right>1 µl</td></tr>
-<tr><td>&nbsp;</td><td align=right>total 50 µl</td></tr>
-</table>
-</td><TD></TD><TD></TD><td>
-<table border="0" width="100px">
-<tr><td align=center>Cycle条件</td></tr>
-</table>
-<table border=1 width="400px">
-<tr><td width="100px" align=center>Pre-Denature</td><td width="100px"  align=center>95°C</td><td width="100px"  align=center>30sec</td></td><td width="100px">&nbsp;</td></tr>
-<tr><td align=center>Denature</td><td align=center>95°C</td><td align=center>30sec</td><td align=center rowspan=3>30 Cyle</td></tr>
-<tr><td align=center>Anneling</td><td align=center>48.6°C</td><td align=center>1min</td></tr>
-<tr><td align=center>Extension</td><td align=center>68°C</td><td align=center>1kb/min</td></tr>
-<tr><td align=center>End</td><td align=center>4°C</td><td align=center>keep</td><td width="100px">&nbsp;</td></tr>
-</table>
-</td></tr>
-</table>
-<BR>
-そしてその後にPCR産物を制限酵素処理することにした<BR>
-制限酵素処理(MLF)<BR>
-MLFのアルカリミニプレップをした後、下表に従って、30分37℃で制限酵素処理を行った。
-<table border=1 width="220px">
-<tr><td width="130px" align=center>ddH<sub>2</sub>O</td><td width="90px"  align=right>2 µl</td></tr>
-<tr><td align=center>BBa_E0240</td><td align=right>50 µl</td></tr>
-<tr><td align=center><em>Eco</em>RⅠ</td><td align=right>1 µl</td></tr>
-<tr><td align=center><em>Pst</em>Ⅰ</td><td align=right>1 µl</td></tr>
-<tr><td align=center>10 x H Buffer</td><td align=right>6 µl</td></tr>
-<tr><td align=center>&nbsp;</td><td align=right>total 60 µl</td></tr>
-</table>
-<BR>
-<BR>
-<BR>
-<BR>
-<BR><BR>
-<BR>
-/8(木)<BR>
-中川<BR>
-(目的)<BR>
-・Flag tag dMLFのゲル抽出
-・pSB1C3のアルカリミニプレップフェノクロ処理、制限酵素処理<BR>
-(方法)<BR>
-MLFのゲル抽出<BR>
-<a herf="http://www.qiagen.com/products/dnacleanup/gelpcrsicleanupsystems/qiaquickgelextractionkit.aspx">QIAquick Gel Extraction Kit</a>を使用した<BR>
-<BR>
-<BR>
-<BR>
-提出用ベクターのアルカリミニプレップ、フェノクロ処理をしたのち乾燥させたDNAに50μlのTEに溶かした<BR>
-そののち制限酵素処理をした<BR>
-<BR>
-制限酵素処理(提出用ベクター)<BR>
-下記の組成に従って反応液を調整した<br>
+==''12th,September''==
-<tr><td><table border=1 width="220px">
+<b>Members</b>
-<tr><td width="130px" align=center>提出用ベクターpSB1C3in ddH<sub>2</sub>O</td><td width="90px"  align=right>50 µl</td></tr>
-<tr><td align=center><em>EcoR</em>Ⅰ</td><td align=right>1 µl</td></tr>
-<tr><td align=center><em>Pst</em>Ⅰ</td><td align=right>1 µl</td></tr>
-<tr><td align=center>10 x H Buffer</td><td align=right>6 µl</td></tr>
-<tr><td align=center>&nbsp;</td><td align=right>total 58 µl</td></tr>
-</table>
 <br>
+:Yoshimura, Nakagawa
+<br>
+:1.We amplified the Flag-MLF by using primer which was made on September 6th.
+:We did PCR reaction with following conditions.
-°Cで20時間静置した<BR>
+:<table border="0"><tr><td>
-<BR>
+:<table border="0" width="150px">
-(結果)<BR>
+:<tr><td align=center>PCR reaction(1)</td></tr>
-MLFを切り出すときにほとんどバンドは見えなかった。<BR>
+:</table>
-そこでまずはベクターとGFPのライゲーションに明日から力を入れることにした。<BR>
+:<table border=1 width="250px">
-<BR><BR>
+:<tr><td width="150px" align=center>10 µM Primer F</td><td width="100px"  align=right>1.5 µl</td></tr>
-<BR>
+:<tr><td align=center>10 µM Primer R</td><td align=right>1.5 µl</td></tr>
-<BR>
+:<tr><td align=center>Template DNA</td><td align=right>1 µl</td></tr>
-<BR>
+:<tr><td align=center>10 x PCR Buffer for KOD Plus</td><td align=right>5 µl</td></tr>
-<BR>
+:<tr><td align=center>dNTPs</td><td align=right>4 µl</td></tr>
-<BR>
+:<tr><td align=center>MgSO<sub>4</sub></td><td align=right>4 µl</td></tr>
-/9★おｋ★<BR>
+:<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>32 µl</td></tr>
-(目的)<BR>
+:<tr><td align=center>KOD Plus</td><td align=right>1 µl</td></tr>
-ベクターとGFPのライゲーションのためのエタノール沈殿<BR>
+:<tr><td>&nbsp;</td><td align=right>total 50 µl</td></tr>
-(方法)<BR>
+:</table>
-まずアドバイザーの方にどうしてライゲーションが失敗するのかを聞いてみた。すると以下の原因が考えられるとなった。<BR>
+:</td><TD></TD><TD></TD><td>
-インサート、ベクターの濃度自体が薄すぎるのではないかという問題<BR>
+:<table border="0" width="100px">
-そもそも抗生物質自体がうまく働いていないのではないか<BR>
+:<tr><td align=center>Cycle</td></tr>
-制限酵素処理が完全にし切れていないのではないか<BR>
+:</table>
-<BR>
+:<table border=1 width="400px">
-そこでエタノール沈殿をすることでベクターとGFPの濃度をあげることによりライゲーションの効率を上げることを画策した<BR>
+:<tr><td width="100px" align=center>Pre-Denature</td><td width="100px"  align=center>94°C</td><td width="100px" align=center>2min</td><td width="100px">&nbsp;</td></tr>
-<BR>
+:<tr><td align=center>Denature</td><td align=center>94°C</td><td align=center>15sec</td><td align=center rowspan=3>35 Cycle</td></tr>
+:<tr><td align=center>Anneling</td><td align=center>55°C</td><td align=center>30sec</td></tr>
-(結果)
+:<tr><td align=center>Extension</td><td align=center>68°C</td><td align=center>1min 20sec</td></tr>
-エタノール沈殿に失敗し沈殿すべきDNAが見当たらず濃度を濃く出来なくなった。<BR>
+:<tr><td align=center>End</td><td align=center>4°C</td><td align=center>keep</td><td width="100px">&nbsp;</td></tr>
-よって再度プレカルチャーの方をあす以降進めていくことになった<BR>
+:</table>
-<BR><BR>
+:</td></tr>
-<BR>
+:</table>
-<BR>
-<BR>
-<BR>
-<BR><BR>
-<p>9/12(月)<br>
 <br>
-吉村、中川<br>
+:<table border="0"><tr><td>
-<br>
+:<table border="0" width="150px">
-<br>
+:<tr><td align=center>PCR reaction(2)</td></tr>
-PCR<br>
+:</table>
-【目的】<br>
+:<table border=1 width="250px">
-/6に作製したプライマーを用いてをFlag-tag dMLFを増幅させる<br>
+:<tr><td width="150px" align=center>10 µM Primer F</td><td width="100px"  align=right>1.5 µl</td></tr>
-<br>
+:<tr><td align=center>10 µM Primer R</td><td align=right>1.5 µl</td></tr>
-【実験方法】<br>
+:<tr><td align=center>Template DNA</td><td align=right>1 µl</td></tr>
-以下の2条件で各1サンプルずつPCRを行った。<BR>
+:<tr><td align=center>10 x PCR Buffer for KOD Plus</td><td align=right>5 µl</td></tr>
-<table border="0"><tr><td>
+:<tr><td align=center>dNTPs</td><td align=right>4 µl</td></tr>
-<table border="0" width="150px">
+:<tr><td align=center>MgSO<sub>4</sub></td><td align=right>2 µl</td></tr>
-<tr><td align=center>PCR条件(1)</td></tr>
+:<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>34 µl</td></tr>
-</table>
+:<tr><td align=center>KOD Plus</td><td align=right>1 µl</td></tr>
-<table border=1 width="220px">
+:<tr><td>&nbsp;</td><td align=right>total 50 µl</td></tr>
-<tr><td width="150px" align=center>10 µM Primer F</td><td width="70px"  align=right>1.5 µl</td></tr>
+:</table>
-<tr><td align=center>10 µM Primer R</td><td align=right>1.5 µl</td></tr>
+:</td><TD></TD><TD></TD><td>
-<tr><td align=center>Template DNA</td><td align=right>1 µl</td></tr>
+:<table border="0" width="100px">
-<tr><td align=center>10 x PCR Buffer for KOD Plus</td><td align=right>5 µl</td></tr>
+:<tr><td align=center>Cycle</td></tr>
-<tr><td align=center>dNTPs</td><td align=right>4 µl</td></tr>
+:</table>
-<tr><td align=center>MgSO<sub>4</sub></td><td align=right>4 µl</td></tr>
+:<table border=1 width="400px">
-<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>32 µl</td></tr>
+:<tr><td width="100px" align=center>Pre-Denature</td><td width="100px"  align=center>94°C</td><td width="100px" align=center>2min</td><td width="100px">&nbsp;</td></tr>
-<tr><td align=center>KOD Plus</td><td align=right>1 µl</td></tr>
+:<tr><td align=center>Denature</td><td align=center>94°C</td><td align=center>15sec</td><td align=center rowspan=3>35 Cycle</td></tr>
-<tr><td>&nbsp;</td><td align=right>total 50 µl</td></tr>
+:<tr><td align=center>Anneling</td><td align=center>55°C</td><td align=center>30sec</td></tr>
-</table>
+:<tr><td align=center>Extension</td><td align=center>68°C</td><td align=center>1min 20sec</td></tr>
-</td><TD></TD><TD></TD><td>
+:<tr><td align=center>End</td><td align=center>4°C</td><td align=center>keep</td><td width="100px">&nbsp;</td></tr>
-<table border="0" width="100px">
+:</table>
-<tr><td align=center>Cycle条件</td></tr>
+:</td></tr>
-</table>
+:</table>
-<table border=1 width="400px">
-<tr><td width="100px" align=center>Pre-Denature</td><td width="100px"  align=center>94°C</td><td width="100px" align=center>2min</td></td><td width="100px">&nbsp;</td></tr>
-<tr><td align=center>Denature</td><td align=center>94°C</td><td align=center>15sec</td><td align=center rowspan=3>35 Cycle</td></tr>
-<tr><td align=center>Anneling</td><td align=center>55°C</td><td align=center>30sec</td></tr>
-<tr><td align=center>Extension</td><td align=center>68°C</td><td align=center>1min 20sec</td></tr>
-<tr><td align=center>End</td><td align=center>4°C</td><td align=center>keep</td><td width="100px">&nbsp;</td></tr>
-</table>
-</td></tr>
-</table>
-<br>
-<table border="0"><tr><td>
-<table border="0" width="150px">
-<tr><td align=center>PCR条件(2)</td></tr>
-</table>
-<table border=1 width="220px">
-<tr><td width="150px" align=center>10 µM Primer F</td><td width="70px"  align=right>1.5 µl</td></tr>
-<tr><td align=center>10 µM Primer R</td><td align=right>1.5 µl</td></tr>
-<tr><td align=center>Template DNA</td><td align=right>1 µl</td></tr>
-<tr><td align=center>10 x PCR Buffer for KOD Plus</td><td align=right>5 µl</td></tr>
-<tr><td align=center>dNTPs</td><td align=right>4 µl</td></tr>
-<tr><td align=center>MgSO<sub>4</sub></td><td align=right>2 µl</td></tr>
-<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>34 µl</td></tr>
-<tr><td align=center>KOD Plus</td><td align=right>1 µl</td></tr>
-<tr><td>&nbsp;</td><td align=right>total 50 µl</td></tr>
-</table>
-</td><TD></TD><TD></TD><td>
-<table border="0" width="100px">
-<tr><td align=center>Cycle条件</td></tr>
-</table>
-<table border=1 width="400px">
-<tr><td width="100px" align=center>Pre-Denature</td><td width="100px"  align=center>94°C</td><td width="100px" align=center>2min</td></td><td width="100px">&nbsp;</td></tr>
-<tr><td align=center>Denature</td><td align=center>94°C</td><td align=center>15sec</td><td align=center rowspan=3>35 Cycle</td></tr>
-<tr><td align=center>Anneling</td><td align=center>55°C</td><td align=center>30sec</td></tr>
-<tr><td align=center>Extension</td><td align=center>68°C</td><td align=center>1min 20sec</td></tr>
-<tr><td align=center>End</td><td align=center>4°C</td><td align=center>keep</td><td width="100px">&nbsp;</td></tr>
-</table>
-</td></tr>
-</table>
-<br>
-各サンプルを回収し、-20゜Cで保存した。
-<br>
-<br>
-【結果】<br>
-アガロースゲル電気泳動に続く
-<br>
-<br>
-<br>
-(目的)<BR>
-大腸菌のプレカルチャー、GFPのPCR,フェノクロ処理、制限酵素処理<BR>
-(方法)<BR>
-昨日全ての素材を失ってしまったのでもう一度両方の素材を作り直すことにした<BR>
-<BR>
-大腸菌をまずは殖菌することにした<BR>
-↓LBプレート(+amp,+kan,+camのいずれか)に大腸菌をまき、37°Cで一晩培養する<BR>
-↓プレートからシングルコロニーを分離する<BR>
-↓2 mlのLB培地で37°Cで一晩振とう培養する<BR>
-<BR>
-GFPの方ももう一度PCRからやり直すことにした<BR>
-<BR>
-PCR
-<table border="0"><tr><td>
-<table border="0" width="150px">
-<tr><td align=center>PCR条件</td></tr>
-</table>
-<table border=1 width="220px">
-<tr><td width="150px" align=center>10 µM Primer F</td><td width="70px"  align=right>1.5 µl</td></tr>
-<tr><td align=center>10 µM Primer R</td><td align=right>1.5 µl</td></tr>
-<tr><td align=center>GFP</td><td align=right>1 µl</td></tr>
-<tr><td align=center>10 x PCR Buffer for KOD Plus</td><td align=right>5 µl</td></tr>
-<tr><td align=center>dNTPs</td><td align=right>4 µl</td></tr>
-<tr><td align=center>MgSO<sub>4</sub></td><td align=right>4 µl</td></tr>
-<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>32 µl</td></tr>
-<tr><td align=center>KOD Plus</td><td align=right>1 µl</td></tr>
-<tr><td>&nbsp;</td><td align=right>total 50 µl</td></tr>
-</table>
-</td><TD></TD><TD></TD><td>
-<table border="0" width="100px">
-<tr><td align=center>Cycle条件</td></tr>
-</table>
-<table border=1 width="400px">
-<tr><td width="100px" align=center>Pre-Denature</td><td width="100px"  align=center>95°C</td><td width="100px"  align=center>30sec</td></td><td width="100px">&nbsp;</td></tr>
-<tr><td align=center>Denature</td><td align=center>95°C</td><td align=center>30sec</td><td align=center rowspan=3>30 Cycle</td></tr>
-<tr><td align=center>Anneling</td><td align=center>48.5°C</td><td align=center>1min</td></tr>
-<tr><td align=center>Extension</td><td align=center>68°C</td><td align=center>1kb/min</td></tr>
-<tr><td align=center>End</td><td align=center>4°C</td><td align=center>keep</td><td width="100px">&nbsp;</td></tr>
-</table>
-</td></tr>
-</table>
-<BR>
-上記の組成に従って混ぜた<BR>
-制限酵素処理(GFP)<BR>
+:We collected each sample and kept them in -20°C.
+:2.Pre-culture of E.coli(DH5α)which contains pSB1C3.
+: I tried doing PCR reaction with following conditions with BBa_E0240 again.
-下記の組成に従って反応液を調整した<BR>
+:<table border="0"><tr><td>
+:<table border="0" width="150px">
+:<tr><td align=center>PCR reaction</td></tr>
+:</table>
+:<table border=1 width="250px">
+:<tr><td width="150px" align=center>10 µM Primer F</td><td width="100px"  align=right>1.5 µl</td></tr>
+:<tr><td align=center>10 µM Primer R</td><td align=right>1.5 µl</td></tr>
+:<tr><td align=center>GFP</td><td align=right>1 µl</td></tr>
+:<tr><td align=center>10 x PCR Buffer for KOD Plus</td><td align=right>5 µl</td></tr>
+:<tr><td align=center>dNTPs</td><td align=right>4 µl</td></tr>
+:<tr><td align=center>MgSO<sub>4</sub></td><td align=right>4 µl</td></tr>
+:<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>32 µl</td></tr>
+:<tr><td align=center>KOD Plus</td><td align=right>1 µl</td></tr>
+:<tr><td>&nbsp;</td><td align=right>total 50 µl</td></tr>
+:</table>
+:</td><TD></TD><TD></TD><td>
+:<table border="0" width="100px">
+:<tr><td align=center>Cycle</td></tr>
+:</table>
+:<table border=1 width="400px">
+:<tr><td width="100px" align=center>Pre-Denature</td><td width="100px"  align=center>95°C</td><td width="100px"  align=center>30sec</td><td width="100px">&nbsp;</td></tr>
+:<tr><td align=center>Denature</td><td align=center>95°C</td><td align=center>30sec</td><td align=center rowspan=3>30 Cycle</td></tr>
+:<tr><td align=center>Anneling</td><td align=center>48.5°C</td><td align=center>1min</td></tr>
+:<tr><td align=center>Extension</td><td align=center>68°C</td><td align=center>1kb/min</td></tr>
+:<tr><td align=center>End</td><td align=center>4°C</td><td align=center>keep</td><td width="100px">&nbsp;</td></tr>
+:</table>
+:</td></tr>
+:</table>
-BBa_E0240のアルカリミニプレップをした後、下表に従って、30分37℃で制限酵素処理を行った。
+: I isolated pSB1C3 by phenol-chloroform treatment .I digested DNA with the following restriction enzymes (30 minutes at 37°C).
-<table border=1 width="220px">
+:<table border=1 width="220px">
-<tr><td width="130px" align=center>ddH<sub>2</sub>O</td><td width="90px"  align=right>2 µl</td></tr>
+:<tr><td width="130px" align=center>ddH<sub>2</sub>O</td><td width="90px"  align=right>2 µl</td></tr>
-<tr><td align=center>BBa_E0240</td><td align=right>50 µl</td></tr>
+:<tr><td align=center>BBa_E0240</td><td align=right>50 µl</td></tr>
-<tr><td align=center><em>Eco</em>RⅠ</td><td align=right>1 µl</td></tr>
+:<tr><td align=center><em>Eco</em>RⅠ</td><td align=right>1 µl</td></tr>
-<tr><td align=center><em>Pst</em>Ⅰ</td><td align=right>1 µl</td></tr>
+:<tr><td align=center><em>Pst</em>Ⅰ</td><td align=right>1 µl</td></tr>
-<tr><td align=center>10 x H Buffer</td><td align=right>6 µl</td></tr>
+:<tr><td align=center>10 x H Buffer</td><td align=right>6 µl</td></tr>
-<tr><td align=center>&nbsp;</td><td align=right>total 60 µl</td></tr>
+:<tr><td align=center>&nbsp;</td><td align=right>total 60 µl</td></tr>
-</table>
+:</table>
+<br>
+<b>Results</b>
+:The E.coli (DH5α) which contains pSB1C3 was increased, but there are some colonies which doesn’t correct color.
+:Possibly deterioration may begin in plate in itself.
+:The DNA of PCR reaction increased next day.
-(結果)<BR>
-大腸菌の方は翌日順調に増えていた、しかし大腸菌の色が明らかに違うものも混じっていた<BR>
-もしかしたらプレート自体に劣化が始まっているのかもしれない<BR>
-PCRにいたっては順調に増えていた<BR>
-<BR>
-<BR><BR>
-<BR>
-<BR>
-/13(火)<br>
+==''13th,September''==
+<b>Member</b>
 <br>
-吉村<br>
+:Yoshimura, Nakagawa
-<br>
-アガロースゲル電気泳動<br>
-【目的】<br>
-PCRで目的のDNAが増幅しているかを調べる。<br>
-<BR>
-【実験方法】<br>
 <br>
-【結果】<br>
+:1.We carried out agarose gel electrophoresis to detect the PCR products.
-泳動後の写真<BR>
+:2.We amplified Flag-tag dMLF by using the primer which I manufactured on 6th September.
-<IMG src="https://static.igem.org/mediawiki/2011/9/9f/2011.9.13%E5%AE%9F%E9%A8%93MLF-PCR%E3%83%81%E3%82%A7%E3%83%83%E3%82%AF.JPG
+: Restriction enzyme processing.
-" width="240px" height="280px" border="0"><BR>
+: This is because after examining a sequence of Flag-tag dMLF, a restriction enzyme site of PstI and XbaI was seen.
+: So, I examined whether PCR product of Flag-tag dMLF is cut by PstI and XbaI.
-PCR条件(1)の方が少しバンドが濃くなっていた。<BR>
-マーカーが流れてしまった。<BR>
-次回からPCR条件(1)の条件でPCRを行う。<BR>
-マーカーが流れてしまったので、次回からは泳動時間を短くして電気泳動を行う。<BR>
-<BR>
-<BR>
-Flag-tag dMLFの制限酵素処理<br>
-【目的】<br>
-Flag-tag dMLFのシーケンスを調べたところ、<em>Pst</em>Ⅰと<em>Xba</em>Ⅰの制限酵素サイトがみられたので、Flag-tag dMLFのPCR産物が途中で<em>Pst</em>Ⅰと<em>Xba</em>Ⅰによって切れてしまわないかを調べる<BR>
-<br>
-【実験方法】<br>
-下表に従って、3サンプル制限酵素処理を行った。<BR>
 (1)<br>
-<tr><td><table border=1 width="220px">
+:<table border=1 width="220px">
-<tr><td width="130px" align=center>MilliQ</td><td width="90px"  align=right>6.5 µl</td></tr>
+:<tr><td width="130px" align=center>MilliQ</td><td width="90px"  align=right>6.5 µl</td></tr>
-<tr><td align=center>Flag-tag dMLF</td><td align=right>20 µl</td></tr>
+:<tr><td align=center>Flag-tag dMLF</td><td align=right>20 µl</td></tr>
-<tr><td align=center><em>Pst</em>Ⅰ</td><td align=right>0.5 µl</td></tr>
+:<tr><td align=center><em>Pst</em>Ⅰ</td><td align=right>0.5 µl</td></tr>
-<tr><td align=center>10 x H Buffer</td><td align=right>3 µl</td></tr>
+:<tr><td align=center>10 x H Buffer</td><td align=right>3 µl</td></tr>
-<tr><td align=center>&nbsp;</td><td align=right>total 30 µl</td></tr>
+:<tr><td align=center>&nbsp;</td><td align=right>total 30 µl</td></tr>
-</table>
+:</table>
 <br>
 (2)<br>
-<tr><td><table border=1 width="220px">
+:<table border=1 width="220px">
-<tr><td width="130px" align=center>MilliQ</td><td width="90px"  align=right>6.5 µl</td></tr>
+:<tr><td width="130px" align=center>MilliQ</td><td width="90px"  align=right>6.5 µl</td></tr>
-<tr><td align=center>Flag-tag dMLF</td><td align=right>20 µl</td></tr>
+:<tr><td align=center>Flag-tag dMLF</td><td align=right>20 µl</td></tr>
-<tr><td align=center><em>Xba</em>Ⅰ</td><td align=right>0.5 µl</td></tr>
+:<tr><td align=center><em>Xba</em>Ⅰ</td><td align=right>0.5 µl</td></tr>
-<tr><td align=center>10 x M Buffer</td><td align=right>3 µl</td></tr>
+:<tr><td align=center>10 x M Buffer</td><td align=right>3 µl</td></tr>
-<tr><td align=center>&nbsp;</td><td align=right>total 30 µl</td></tr>
+:<tr><td align=center>&nbsp;</td><td align=right>total 30 µl</td></tr>
-</table>
+:</table>
 <br>
 (3)<br>
-<tr><td><table border=1 width="220px">
+:<table border=1 width="220px">
-<tr><td width="130px" align=center>MilliQ</td><td width="90px"  align=right>6 µl</td></tr>
+:<tr><td width="130px" align=center>MilliQ</td><td width="90px"  align=right>6 µl</td></tr>
-<tr><td align=center>Flag-tag dMLF</td><td align=right>20 µl</td></tr>
+:<tr><td align=center>Flag-tag dMLF</td><td align=right>20 µl</td></tr>
-<tr><td align=center><em>Pst</em>Ⅰ</td><td align=right>0.5 µl</td></tr>
+:<tr><td align=center><em>Pst</em>Ⅰ</td><td align=right>0.5 µl</td></tr>
-<tr><td align=center><em>Xba</em>Ⅰ</td><td align=right>0.5 µl</td></tr>
+:<tr><td align=center><em>Xba</em>Ⅰ</td><td align=right>0.5 µl</td></tr>
-<tr><td align=center>10 x M Buffer</td><td align=right>3 µl</td></tr>
+:<tr><td align=center>10 x M Buffer</td><td align=right>3 µl</td></tr>
-<tr><td align=center>&nbsp;</td><td align=right>total 30 µl</td></tr>
+:<tr><td align=center>&nbsp;</td><td align=right>total 30 µl</td></tr>
-</table>
+:</table>
-<br>
+:After incubate it in 37°C for 50 minutes, I performed electrophoresis with agarose gel.
+:Photograph after the electrophoresis.
-゜Cで50分間インキュベートした後、アガロースゲル電気泳動を行った。<br>
+:3.To amplify Flag-tag dMLF cDNA, PCR reactions were carried out under the following conditions.
-<BR>
-<br>
-【結果】<br>
+:<table border="0"><tr><td>
-泳動後の写真<BR>
+:<table border="0" width="150px">
+:<tr><td align=center>PCR reaction</td></tr>
+:</table>
+:<table border=1 width="250px">
+:<tr><td width="150px" align=center>10 µM Primer F</td><td width="100px"  align=right>1.5 µl</td></tr>
+:<tr><td align=center>10 µM Primer R</td><td align=right>1.5 µl</td></tr>
+:<tr><td align=center>Template DNA</td><td align=right>1 µl</td></tr>
+:<tr><td align=center>10 x PCR Buffer for KOD Plus</td><td align=right>5 µl</td></tr>
+:<tr><td align=center>dNTPs</td><td align=right>4 µl</td></tr>
+:<tr><td align=center>MgSO<sub>4</sub></td><td align=right>4 µl</td></tr>
+:<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>32 µl</td></tr>
+:<tr><td align=center>KOD Plus</td><td align=right>1 µl</td></tr>
+:<tr><td>&nbsp;</td><td align=right>total 50 µl</td></tr>
+:</table>
+:</td><TD></TD><TD></TD><td>
+:<table border="0" width="100px">
+:<tr><td align=center>Cycle</td></tr>
+:</table>
+:<table border=1 width="400px">
+:<tr><td width="100px" align=center>Pre-Denature</td><td width="100px"  align=center>94°C</td><td width="100px" align=center>2min</td><td width="100px">&nbsp;</td></tr>
+:<tr><td align=center>Denature</td><td align=center>94°C</td><td align=center>15sec</td><td align=center rowspan=3>35 Cycle</td></tr>
+:<tr><td align=center>Anneling</td><td align=center>55°C</td><td align=center>30sec</td></tr>
+:<tr><td align=center>Extension</td><td align=center>68°C</td><td align=center>1min 20sec</td></tr>
+:<tr><td align=center>End</td><td align=center>4°C</td><td align=center>keep</td><td width="100px">&nbsp;</td></tr>
+:</table>
+:</td></tr>
+:</table>
+:We collected each sample and kept them in -20°C.
+<br>
+<b>Results</b>
+:1.Image of the agarose gel.
+:<html><body>
+<IMG src="https://static.igem.org/mediawiki/2011/9/99/2011.09.13_%E4%B8%AD%E5%B7%9D.JPG
+" width="240px" height="280px" border="0">
+</body></html>
+<br>
+:2.Images of the agarose gel.
+:
+:<html><body>
 <IMG src="https://static.igem.org/mediawiki/2011/8/8a/2011.09.13MLF%E3%83%81%E3%82%A7%E3%83%83%E3%82%AFPst.Xba.JPG
 " width="240px" height="280px" border="0">
-<IMG src="https://static.igem.org/mediawiki/2011/d/dc/2011.09.13MLF%E3%83%81%E3%82%A7%E3%83%83%E3%82%AFXba.JPG
-" width="240px" height="280px" border="0"><BR>
-Flag-tag dMLFは<em>Pst</em>Ⅰと<em>Xba</em>Ⅰの制限酵素処理によって切れることはなかった。<BR>
-マーカーが開ききっていなかった。<BR>
 <BR>
+:<IMG src="https://static.igem.org/mediawiki/2011/d/dc/2011.09.13MLF%E3%83%81%E3%82%A7%E3%83%83%E3%82%AFXba.JPG
+" width="240px" height="280px" border="0">
+</body></html>
+:Flag-tag dMLF aren’t cut by restriction enzyme of PstI and XbaI.
+:A marker did not enough to drift.
+<br>
-【考察】<BR>
-今回の実験ではFlag-tag dMLFは<em>Pst</em>Ⅰと<em>Xba</em>Ⅰの制限酵素処理によって切れることはなかったが、制限酵素処理時間が短いという可能性があるため、制限酵素処理時間を長くして再度実験を行う。<BR>
-また、マーカーのラダーが開ききっていないため、泳動時間が短かったと考えられる。<BR>
-次回はゲルの濃度を2倍にして電気泳動を行う。<BR>
-<BR>
-PCR<br>
+==''14th,September''==
-【目的】<br>
+<b>Members</b>
-/6に作製したプライマーを用いてFlag-tag dMLFを増幅させる<br>
 <br>
-【実験方法】<br>
+:Yoshimura
-以下の条件で4サンプルPCRを行った。<BR>
-<table border="0"><tr><td>
-<table border="0" width="150px">
-<tr><td align=center>PCR条件</td></tr>
-</table>
-<table border=1 width="220px">
-<tr><td width="150px" align=center>10 µM Primer F</td><td width="70px"  align=right>1.5 µl</td></tr>
-<tr><td align=center>10 µM Primer R</td><td align=right>1.5 µl</td></tr>
-<tr><td align=center>Template DNA</td><td align=right>1 µl</td></tr>
-<tr><td align=center>10 x PCR Buffer for KOD Plus</td><td align=right>5 µl</td></tr>
-<tr><td align=center>dNTPs</td><td align=right>4 µl</td></tr>
-<tr><td align=center>MgSO<sub>4</sub></td><td align=right>4 µl</td></tr>
-<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>32 µl</td></tr>
-<tr><td align=center>KOD Plus</td><td align=right>1 µl</td></tr>
-<tr><td>&nbsp;</td><td align=right>total 50 µl</td></tr>
-</table>
-</td><TD></TD><TD></TD><td>
-<table border="0" width="100px">
-<tr><td align=center>Cycle条件</td></tr>
-</table>
-<table border=1 width="400px">
-<tr><td width="100px" align=center>Pre-Denature</td><td width="100px"  align=center>94°C</td><td width="100px" align=center>2min</td></td><td width="100px">&nbsp;</td></tr>
-<tr><td align=center>Denature</td><td align=center>94°C</td><td align=center>15sec</td><td align=center rowspan=3>35 Cycle</td></tr>
-<tr><td align=center>Anneling</td><td align=center>55°C</td><td align=center>30sec</td></tr>
-<tr><td align=center>Extension</td><td align=center>68°C</td><td align=center>1min 20sec</td></tr>
-<tr><td align=center>End</td><td align=center>4°C</td><td align=center>keep</td><td width="100px">&nbsp;</td></tr>
-</table>
-</td></tr>
-</table>
 <br>
-各サンプルを回収し、-20゜Cで保存した。
+:1.We carried out agarose gel electrophoresis to detect the PCR products.
+:2.I extracted GFP, DNA of the vector from gel using  [http://www.qiagen.com/products/dnacleanup/gelpcrsicleanupsystems/qiaquickgelextractionkit.aspx|QIAquick Gel Extraction Kit].
 <br>
+<b>Results</b>
+.Image of the agarose gel.
 <br>
-【結果】<br>
+:<html><body>
-アガロースゲル電気泳動に続く
+<IMG src="https://static.igem.org/mediawiki/2011/5/53/2011.09.14MLF-PCR%E3%83%81%E3%82%A7%E3%83%83%E3%82%AF.JPG
+" width="240px" height="280px" border="0">
+</body></html>
 <br>
-<br>
+:After doubling the density of gel, a marker did not drift.
-<br>
+:I will decide to perform it with 2% of agarose gel about the electrophoresis of Flag-tag MLF in future.
-/13<BR>
+.I measured the density with an absorbance meter afterwards.
-(目的)<BR>
-再度GFPのPCR、ベクターのアルカリミニプレップ、フェノクロ処理、制限酵素処理<BR>
-(方法)<BR>
-昨日行ったGFPのPCRを行った量ではこの後の失敗が起こった時に心もとないということで保管する名目で再度PCRを行った<BR>
-<BR>
-PCR
-<table border="0"><tr><td>
-<table border="0" width="150px">
-<tr><td align=center>PCR条件</td></tr>
-</table>
-<table border=1 width="220px">
-<tr><td width="150px" align=center>10 µM Primer F</td><td width="70px"  align=right>1.5 µl</td></tr>
-<tr><td align=center>10 µM Primer R</td><td align=right>1.5 µl</td></tr>
-<tr><td align=center>GFP</td><td align=right>1 µl</td></tr>
-<tr><td align=center>10 x PCR Buffer for KOD Plus</td><td align=right>5 µl</td></tr>
-<tr><td align=center>dNTPs</td><td align=right>4 µl</td></tr>
-<tr><td align=center>MgSO<sub>4</sub></td><td align=right>4 µl</td></tr>
-<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>32 µl</td></tr>
-<tr><td align=center>KOD Plus</td><td align=right>1 µl</td></tr>
-<tr><td>&nbsp;</td><td align=right>total 50 µl</td></tr>
-</table>
-</td><TD></TD><TD></TD><td>
-<table border="0" width="100px">
-<tr><td align=center>Cycle条件</td></tr>
-</table>
-<table border=1 width="400px">
-<tr><td width="100px" align=center>Pre-Denature</td><td width="100px"  align=center>95°C</td><td width="100px"  align=center>30sec</td></td><td width="100px">&nbsp;</td></tr>
-<tr><td align=center>Denature</td><td align=center>95°C</td><td align=center>30sec</td><td align=center rowspan=3>30 Cycle</td></tr>
-<tr><td align=center>Anneling</td><td align=center>48.5°C</td><td align=center>1min</td></tr>
-<tr><td align=center>Extension</td><td align=center>68°C</td><td align=center>1kb/min</td></tr>
-<tr><td align=center>End</td><td align=center>4°C</td><td align=center>keep</td><td width="100px">&nbsp;</td></tr>
-</table>
-</td></tr>
-</table>
-<BR>
-上記の組成に従って混ぜた<BR>
-制限酵素処理(GFP)<BR>
+:GFP
+:<table border=1 width="160px">
-下記の組成に従って反応液を調整した<BR>
+:<tr><td width="70px" align=center>1</td><td width="90px"  align=right>0.158</td></tr>
+:<tr><td align=center>2</td><td align=right>0.147</td></tr>
-BBa_E0240のアルカリミニプレップをした後、下表に従って、30分37℃で制限酵素処理を行った。
+:<tr><td align=center>3</td><td align=right>0.160</td></tr>
+:<tr><td align=center>4</td><td align=right>0.159</td></tr>
-<table border=1 width="220px">
+:<tr><td align=center>5</td><td align=right>0.158</td></tr>
-<tr><td width="130px" align=center>ddH<sub>2</sub>O</td><td width="90px"  align=right>2 µl</td></tr>
+:<tr><td align=center>ave.</td><td align=right>0.156</td></tr>
-<tr><td align=center>BBa_E0240</td><td align=right>50 µl</td></tr>
+:</table>
-<tr><td align=center><em>Eco</em>RⅠ</td><td align=right>1 µl</td></tr>
+:pSB1C3<BR>
-<tr><td align=center><em>Pst</em>Ⅰ</td><td align=right>1 µl</td></tr>
+:<table border=1 width="160px">
-<tr><td align=center>10 x H Buffer</td><td align=right>6 µl</td></tr>
+:<tr><td width="70px" align=center>1</td><td width="90px"  align=right>0.029</td></tr>
-<tr><td align=center>&nbsp;</td><td align=right>total 60 µl</td></tr>
+:<tr><td align=center>2</td><td align=right>0.021</td></tr>
-</table>
+:<tr><td align=center>3</td><td align=right>0.028</td></tr>
-<BR>
+:<tr><td align=center>4</td><td align=right>0.022</td></tr>
-次に同時進行でベクターの方は菌が増えていたのでアルカリミニプレップ、フェノクロ処理、制限酵素処理を行った
+:<tr><td align=center>5</td><td align=right>0.025</td></tr>
+:<tr><td align=center>ave.</td><td align=right>0.025</td></tr>
-フェノクロ処理後は
+:</table>
-<BR>
-制限酵素処理(ベクター)<BR>
-pSB1C3のアルカリミニプレップをした後、下表に従って、30分37℃で制限酵素処理を行った。
-<table border=1 width="220px">
-<tr><td width="130px" align=center>ddH<sub>2</sub>O</td><td width="90px"  align=right>2 µl</td></tr>
-<tr><td align=center>pSB1C3</td><td align=right>50 µl</td></tr>
-<tr><td align=center><em>Eco</em>RⅠ</td><td align=right>1 µl</td></tr>
-<tr><td align=center><em>Pst</em>Ⅰ</td><td align=right>1 µl</td></tr>
-<tr><td align=center>10 x H Buffer</td><td align=right>6 µl</td></tr>
-<tr><td align=center>&nbsp;</td><td align=right>total 60 µl</td></tr>
-</table>
-<BR>
-(結果)<BR>
-GFPの方は昨日同様に成功しているといえた、そしてベクターの方も順調に増えていたので無事にDNAを回収できた<BR>
-<BR>
-<BR>
-<BR>
-<BR>
-/14(水)<BR>
-<BR>
-吉村<BR>
-<BR>
-アガロースゲル電気泳動<br>
-【目的】<br>
-PCRで目的のDNAが増幅しているかを調べる。<br>
-<BR>
 <br>
-【結果】<br>
+:GFP<BR>
-泳動後の写真<BR>
+:<HTML><BODY>
-<IMG src="https://static.igem.org/mediawiki/2011/5/53/2011.09.14MLF-PCR%E3%83%81%E3%82%A7%E3%83%83%E3%82%AF.JPG
-" width="240px" height="280px" border="0"><BR>
-ゲルの濃度を2倍にしたところ、マーカーが流れることがなかった。<BR>
-Flag-tag MLFの電気泳動に関しては今後、2%のアガロースゲルで行うこととする。<BR>
-<BR>
-<BR>
-/14<BR>
-(目的)<BR>
-GFP、提出用ベクターのゲル抽出<BR>
-(方法)<BR>
-<BR>
-<a herf="http://www.qiagen.com/products/dnacleanup/gelpcrsicleanupsystems/qiaquickgelextractionkit.aspx">QIAquick Gel Extraction Kit</a>を使用してゲルからGFP、ベクターのDNAを抽出した。<BR>
-<BR>
-その後吸光度計を使って濃度の測定をした。<BR>
-<BR>
-GFPの濃度測定<BR>
-<tr><td><table border=1 width="160px">
-<tr><td width="70px" align=center>1回目</td><td width="90px"  align=right>0.158</td></tr>
-<tr><td align=center>2回目</td><td align=right>0.147</td></tr>
-<tr><td align=center>3回目</td><td align=right>0.160</td></tr>
-<tr><td align=center>4回目</td><td align=right>0.159</td></tr>
-<tr><td align=center>5回目</td><td align=right>0.158</td></tr>
-<tr><td align=center>ave.</td><td align=right>0.156</td></tr>
-</table>
-提出用ベクターの濃度測定<BR>
-<tr><td><table border=1 width="160px">
-<tr><td width="70px" align=center>1回目</td><td width="90px"  align=right>0.029</td></tr>
-<tr><td align=center>2回目</td><td align=right>0.021</td></tr>
-<tr><td align=center>3回目</td><td align=right>0.028</td></tr>
-<tr><td align=center>4回目</td><td align=right>0.022</td></tr>
-<tr><td align=center>5回目</td><td align=right>0.025</td></tr>
-<tr><td align=center>ave.</td><td align=right>0.025</td></tr>
-</table>
-左、GFPのゲル抽出写真　右、ベクターのゲル抽出写真<BR>
 <IMG src="https://static.igem.org/mediawiki/2011/3/3d/2011.09.14_%E4%B8%AD%E5%B7%9D.JPG
 " width="240px" height="280px" border="0">
+</BODY></HTML>
+:pSB1C3
+:<HTML><BODY>
 <IMG src="https://static.igem.org/mediawiki/2011/3/3a/2011.09.26_%E3%83%99%E3%82%AF%E3%82%BF%E3%83%BC%E3%82%B2%E3%83%AB%E6%8A%BD%E5%87%BA%282%29.JPG
-" width="240px" height="280px" border="0"><BR>
+" width="240px" height="280px" border="0">
+</BODY></HTML>
+<br>
+:The BBa_E0240 succeeded in the extraction from gel and recorded 156ng/µl by the measurement with the absorbance meter and the pSB1C3 became 25ng/µl.
+:Therefore I decided to begin an experiment of the ligation again from the next day.
-(結果)<BR>
+==''17th,September''==
-ゲルからの抽出はGFPは成功して吸光度計での測定で156ng/µlを記録した、そしてベクターの方は薄いながらも25ng/μlとなり材料がそろった<BR>
+<b>Members</b>
-そこで翌日から再度ライゲーションの実験に入ることにした<BR>
+<br>
+:Nakagawa
+<br>
+:1.Ligate with BBa_E0240 and pSB1C3
+:2.Pre-culture and the alkaline-lysis method of pSB1C3
+<br>
+:I adjusted reaction liquid according to the following composition.
+pSB1C3:GFP＝1:2<BR>
+:<table border=1 width="200px">
+:<tr><td width="130px" align=center>insert</td><td width="70px" align=right>0.3 µl</td></tr>
+:<tr><td align=center>vector</td><td align=right>2.0µl</td></tr>
+:<tr><td align=center>10 x Buffer</td><td align=right>2.5 µl</td></tr>
+:<tr><td align=center>F4 ligase</td><td align=right>0.5 µl</td></tr>
+:<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>1.7 µl</td></tr>
+:<tr><td>&nbsp;</td><td align=right>total 7 µl</td></tr>
+:</table>
+pSB1C3:GFP＝1:5<BR>
+:<table border=1 width="200px">
+:<tr><td width="130px" align=center>insert</td><td width="70px" align=right>1.0 µl</td></tr>
+:<tr><td align=center>vector</td><td align=right>1.0 µl</td></tr>
+:<tr><td align=center>10 x Buffer</td><td align=right>2.5 µl</td></tr>
+:<tr><td align=center>F4 ligase</td><td align=right>0.5 µl</td></tr>
+:<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>2.0 µl</td></tr>
+:<tr><td>&nbsp;</td><td align=right>total 7 µl</td></tr>
+:</table>
+pSB1C3:GFP＝1:10<BR>
+:<table border=1 width="200px">
+:<tr><td width="130px" align=center>insert</td><td width="70px" align=right>1.3 µl</td></tr>
+:<tr><td align=center>vector</td><td align=right>1.0 µl</td></tr>
+:<tr><td align=center>10 x Buffer</td><td align=right>2.5 µl</td></tr>
+:<tr><td align=center>F4 ligase</td><td align=right>0.5 µl</td></tr>
+:<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>1.7 µl</td></tr>
+:<tr><td>&nbsp;</td><td align=right>total 7 µl</td></tr>
+:</table>
+:I incubated them in 16°C for 30 min.
+:After that, I transformed them, and I poured them into 300 µl of SOC medium and add them on the plate after culture for 30 minutes.
-<BR>
+:I thought that I tried to pick at only a red colony as a point to keep in mind of this time and lowered the possibility of contaminating it.
-/15<BR>
+<br>
-(目的)<BR>
+<b>Results</b>
-GFP、ベクターのライゲーションとベクターのプレカルチャー<BR>
+:Here are many colonies on the LB medium.
-(方法)<BR>
+:Possibly contaminating is thought for example an antibiotic does not work.
-それぞれの濃度は156ng/µlと25 ng/µlだった。<BR>
+:The pSB1C3 was not able to be refined again.
-そこで先の失敗より濃度の割合を変えて下記の組成に従って反応液を調整した<BR>
-提出用ベクター:GFP＝1:2<BR>
-<table border=1 width="200px">
-<tr><td width="130px" align=center>insert</td><td width="70px" align=right>0.3 µl</td></tr>
-<tr><td align=center>vector</td><td align=right>2.0µl</td></tr>
-<tr><td align=center>10 x Buffer</td><td align=right>2.5 µl</td></tr>
-<tr><td align=center>F4 ligase</td><td align=right>0.5 µl</td></tr>
-<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>1.7 µl</td></tr>
-<tr><td>&nbsp;</td><td align=right>total 7 µl</td></tr>
-</table>
-提出用ベクター:GFP＝1:5<BR>
-<table border=1 width="200px">
-<tr><td width="130px" align=center>insert</td><td width="70px" align=right>1.0 µl</td></tr>
-<tr><td align=center>vector</td><td align=right>1.0 µl</td></tr>
-<tr><td align=center>10 x Buffer</td><td align=right>2.5 µl</td></tr>
-<tr><td align=center>F4 ligase</td><td align=right>0.5 µl</td></tr>
-<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>2.0 µl</td></tr>
-<tr><td>&nbsp;</td><td align=right>total 7 µl</td></tr>
-</table>
-提出用ベクター:GFP＝1:10<BR>
-<table border=1 width="200px">
-<tr><td width="130px" align=center>insert</td><td width="70px" align=right>1.3 µl</td></tr>
-<tr><td align=center>vector</td><td align=right>1.0 µl</td></tr>
-<tr><td align=center>10 x Buffer</td><td align=right>2.5 µl</td></tr>
-<tr><td align=center>F4 ligase</td><td align=right>0.5 µl</td></tr>
-<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>1.7 µl</td></tr>
-<tr><td>&nbsp;</td><td align=right>total 7 µl</td></tr>
-</table>
-<BR>
-°C、30minでincubateした<BR>
-ライゲーション後、形質転換を行うためにトラフォを行った。<BR>
-(結果)<BR>
+==''19th,September''==
-ライゲーションに関しては翌日のコロニーの育成は見られなかった<BR>
+<b>Members</b>
-プレカルチャーした大腸菌は数本赤色残り白色といった色の違いが出来ていたが無事に増えて濁っていた<BR>
+<br>
+:Yoshimura Nakagawa
+<br>
+:1.We transformed and performed restriction enzyme processing
+:2.Density check of BBa_E0240 and pSB1C3
+:3.Pre-culture with yesterday colony and pSB1C3 to the new LB plate
+<br>
+:I digested with the ''Xho''I according to the following composition.
-/16<BR>
+:(1)<br>
-(目的)<BR>
+:<table border=1 width="220px">
-GFPとベクターのライゲーション、育成した大腸菌のアルカリミニプレップ、プレカルチャー<BR>
+:<tr><td width="130px" align=center>MilliQ</td><td width="90px"  align=right>6.5 µl</td></tr>
-(方法)<BR>
+:<tr><td align=center>Flag-tag dMLF</td><td align=right>20 µl</td></tr>
-それぞれの濃度は156ng/µlと25 ng/µlだった。<BR>
+:<tr><td align=center><em>Pst</em>Ⅰ</td><td align=right>0.5 µl</td></tr>
+:<tr><td align=center>10 x H Buffer</td><td align=right>3 µl</td></tr>
+:<tr><td align=center>&nbsp;</td><td align=right>total 30 µl</td></tr>
+:</table>
+<br>
+:(2)<br>
+:<table border=1 width="220px">
+:<tr><td width="130px" align=center>MilliQ</td><td width="90px"  align=right>6.5 µl</td></tr>
+:<tr><td align=center>Flag-tag dMLF</td><td align=right>20 µl</td></tr>
+:<tr><td align=center><em>Xba</em>Ⅰ</td><td align=right>0.5 µl</td></tr>
+:<tr><td align=center>10 x M Buffer</td><td align=right>3 µl</td></tr>
+:<tr><td align=center>&nbsp;</td><td align=right>total 30 µl</td></tr>
+:</table>
+<br>
-そして先の失敗より濃度の割合を変えるだけでなく回復培養を行った。
+:(3)<br>
-下記の組成に従って反応液を調整した<BR>
+:<table border=1 width="220px">
-提出用ベクター:GFP＝1:2<BR>
+:<tr><td width="130px" align=center>MilliQ</td><td width="90px"  align=right>6 µl</td></tr>
-<table border=1 width="200px">
+:<tr><td align=center>Flag-tag dMLF</td><td align=right>20 µl</td></tr>
-<tr><td width="130px" align=center>insert</td><td width="70px" align=right>0.3 µl</td></tr>
+:<tr><td align=center><em>Pst</em>Ⅰ</td><td align=right>0.5 µl</td></tr>
-<tr><td align=center>vector</td><td align=right>2.0µl</td></tr>
+:<tr><td align=center><em>Xba</em>Ⅰ</td><td align=right>0.5 µl</td></tr>
-<tr><td align=center>10 x Buffer</td><td align=right>2.5 µl</td></tr>
+:<tr><td align=center>10 x M Buffer</td><td align=right>3 µl</td></tr>
-<tr><td align=center>F4 ligase</td><td align=right>0.5 µl</td></tr>
+:<tr><td align=center>&nbsp;</td><td align=right>total 30 µl</td></tr>
-<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>1.7 µl</td></tr>
+:</table>
-<tr><td>&nbsp;</td><td align=right>total 7 µl</td></tr>
-</table>
-提出用ベクター:GFP＝1:5<BR>
-<table border=1 width="200px">
-<tr><td width="130px" align=center>insert</td><td width="70px" align=right>1.0 µl</td></tr>
-<tr><td align=center>vector</td><td align=right>1.0 µl</td></tr>
-<tr><td align=center>10 x Buffer</td><td align=right>2.5 µl</td></tr>
-<tr><td align=center>F4 ligase</td><td align=right>0.5 µl</td></tr>
-<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>2.0 µl</td></tr>
-<tr><td>&nbsp;</td><td align=right>total 7 µl</td></tr>
-</table>
-提出用ベクター:GFP＝1:10<BR>
-<table border=1 width="200px">
-<tr><td width="130px" align=center>insert</td><td width="70px" align=right>1.3 µl</td></tr>
-<tr><td align=center>vector</td><td align=right>1.0 µl</td></tr>
-<tr><td align=center>10 x Buffer</td><td align=right>2.5 µl</td></tr>
-<tr><td align=center>F4 ligase</td><td align=right>0.5 µl</td></tr>
-<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>1.7 µl</td></tr>
-<tr><td>&nbsp;</td><td align=right>total 7 µl</td></tr>
-</table>
-<BR>
-°C、30minでincubateした<BR>
-ライゲーション後、、形質転換を行い、その後にSOC培地300μlに注入し30分培養後プレートにまいた<BR>
+:After incubate it in 37 ゜ C for 18 hours, I performed agarose gel electrophoresis.
+<br>
+:Checking the amount of the purified GFP,
+:<table border=1 width="160px">
+:<tr><td width="70px" align=center>1 x </td><td width="90px"  align=right>1µl</td></tr>
+:<tr><td align=center>2 x </td><td align=right>1µl</td></tr>
+:<tr><td align=center>4 x </td><td align=right>1µl</td></tr>
+:<tr><td align=center>8 x </td><td align=right>1µl</td></tr>
+:</table><BR>
+:Image of agarose gel after DNA fragment isolation for Checking the amount of the purified GFP.
+<br>
+:<HTML><BODY>
+<IMG src="https://static.igem.org/mediawiki/2011/5/52/2011.09.19_GFP%E6%BF%83%E5%BA%A6%E3%83%81%E3%82%A7%E3%83%83%E3%82%AF.JPG" width="250px" height="250px" border="0">
+</BODY></HTML>
+<br>
+<b>Results</b>
+:The increase of BBa_E0240 was good, and upbringing of the big colony was seen.
+:pSB1C3 was not BBa_E0240 too, but was all right because some number grew.
-(結果)<BR>
-GFPのコロニーの育成はほとんど見られなかった<BR>
-大腸菌は途中でDNAが消えてしまい精製することが不可能になった<BR><BR><BR>
-<BR>
-/17(土)<BR>
-<BR>
-吉村・横井川<BR>
-<BR>
-ライゲーション<BR>
-<BR>
-/17<BR>
-(目的)<BR>
-GFPとベクターのライゲーション、アルカリミニプレップ、プレカルチャー<BR>
-(方法)<BR>
-下記の組成に従って反応液を調整した<BR>
-提出用ベクター:GFP＝1:2<BR>
-<table border=1 width="200px">
-<tr><td width="130px" align=center>insert</td><td width="70px" align=right>0.3 µl</td></tr>
-<tr><td align=center>vector</td><td align=right>2.0µl</td></tr>
-<tr><td align=center>10 x Buffer</td><td align=right>2.5 µl</td></tr>
-<tr><td align=center>F4 ligase</td><td align=right>0.5 µl</td></tr>
-<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>1.7 µl</td></tr>
-<tr><td>&nbsp;</td><td align=right>total 7 µl</td></tr>
-</table>
-提出用ベクター:GFP＝1:5<BR>
-<table border=1 width="200px">
-<tr><td width="130px" align=center>insert</td><td width="70px" align=right>1.0 µl</td></tr>
-<tr><td align=center>vector</td><td align=right>1.0 µl</td></tr>
-<tr><td align=center>10 x Buffer</td><td align=right>2.5 µl</td></tr>
-<tr><td align=center>F4 ligase</td><td align=right>0.5 µl</td></tr>
-<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>2.0 µl</td></tr>
-<tr><td>&nbsp;</td><td align=right>total 7 µl</td></tr>
-</table>
-提出用ベクター:GFP＝1:10<BR>
-<table border=1 width="200px">
-<tr><td width="130px" align=center>insert</td><td width="70px" align=right>1.3 µl</td></tr>
-<tr><td align=center>vector</td><td align=right>1.0 µl</td></tr>
-<tr><td align=center>10 x Buffer</td><td align=right>2.5 µl</td></tr>
-<tr><td align=center>F4 ligase</td><td align=right>0.5 µl</td></tr>
-<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>1.7 µl</td></tr>
-<tr><td>&nbsp;</td><td align=right>total 7 µl</td></tr>
-</table>
-°C、30minでincubateした
-<BR>
-ライゲーション後、、形質転換を行い、その後にSOC培地300μlに注入し30分培養後プレートにまいた<BR
-大腸菌のベクターのアルカリミニプレップとプレカルチャーを行った<BR>
-このときの留意点として赤いコロニーのみつつくことを心がけてコンタミの可能性を下げることを考えた<BR>
-(結果)<BR>
-今度はライゲーション産物におかしな量のコロニーの育成が見られた、もしかしたらコンタミの可能性があると考えられる（抗生物質が効いていない）、ベクターはまた精製することが出来なかった。<BR>
-/18<BR>
-(目的)<BR>
-プレートチェックをすることにした、ベクターの精製をまたすることにした<BR>
-(方法)<BR>
-pSB1C3のアルカリミニプレップをした<BR>
-プレートチェックのためにコンピテントセルのみを今まで使っていた培地にまいて増えるかどうかを見てみることにした<BR>
+==''24th,September''==
+<b>Members</b>
+<br>
+:Yoshimura Nakagawa
+<br>
+:1.alkaline-lysis method, phenol-chloroform treatment
+:2.restriction enzymes, extraction with pSB1C3
+:3.restriction enzymes processing with BBa_E0240
-(結果)<BR>
+:Yield rose markedly when I exchanged isopropanol.
-プレート自体がおかしくなっているのではないかという推測は間違ってはおらず<BR>
+:After alkaline-lysis method, phenol-chloroform treatment with pSB1C3,restriction enzymes processing with BBa_E0240 and pSB1C3
-プレートに生えるはずのないコロニーが出てきているのもあった。<BR>
+:According to a list shown below, I performed restriction enzyme processing at 37 overnight.
-また新しくプレートを明日から作り直すことにした<BR>
+:<table border=1 width="220px">
+:<tr><td width="130px" align=center>ddH<sub>2</sub>O</td><td width="90px"  align=right>2 µl</td></tr>
+:<tr><td align=center>BBa_E0240</td><td align=right>50 µl</td></tr>
+:<tr><td align=center><em>Eco</em>RⅠ</td><td align=right>1 µl</td></tr>
+:<tr><td align=center><em>Pst</em>Ⅰ</td><td align=right>1 µl</td></tr>
+:<tr><td align=center>10 x H Buffer</td><td align=right>6 µl</td></tr>
+:<tr><td align=center>&nbsp;</td><td align=right>total 60 µl</td></tr>
+:</table>
-<BR>
+:The restriction enzyme-digested pSB1C3 were extracted from the agarose gel by QIAquick Gel Extraction Kit.
-<BR>
+:Photograph after the extraction.
-/19(月)
-<BR>
-トランスフォーメーション<BR>
-<BR>
-【目的】<BR>
-<BR>
-<BR>
-【実験方法】
-↓氷上でコンピテント細胞(XL1-Blue:大腸菌株)を解凍した<BR>
-↓前もって冷やしておいた1.5 mlチューブに100 µlのコンピテント細胞を分注した<BR>
-↓余ったコンピテント細胞は-80°Cの冷凍庫に戻した<BR>
-↓DNAをチューブに1～5 µl加えて、氷上で30分間冷やす<BR>
-↓42°Cで45秒間熱ショックを与えた<BR>
-↓0.9 mlのSOC培地を加えた<BR>
-↓37°Cで1時間、振とう培養した<BR>
-↓1 mlをLBプレート(+amp,+kan,+camのいずれか)にまいた<BR>
-↓37°Cで一晩培養した<BR>
-<BR>
-【結果】<BR>
-<BR>
-<BR>
-<BR>
-制限酵素処理<BR>
-【目的】<BR>
-Flag-tag dMLFのシーケンスを調べたところ、PstⅠとXbaⅠの制限酵素サイトがみられたので、Flag-tag dMLFのPCR産物が途中でPstⅠとXbaⅠによって切れてしまわないかを調べる。<BR>
-<BR>
-【実験方法】<BR>
-下表に従って、3サンプル制限酵素処理を行った。<BR>
-(1)<br>
-<tr><td><table border=1 width="220px">
-<tr><td width="130px" align=center>MilliQ</td><td width="90px"  align=right>6.5 µl</td></tr>
-<tr><td align=center>Flag-tag dMLF</td><td align=right>20 µl</td></tr>
-<tr><td align=center><em>Pst</em>Ⅰ</td><td align=right>0.5 µl</td></tr>
-<tr><td align=center>10 x H Buffer</td><td align=right>3 µl</td></tr>
-<tr><td align=center>&nbsp;</td><td align=right>total 30 µl</td></tr>
-</table>
 <br>
+<b>Results</b>
-(2)<br>
-<tr><td><table border=1 width="220px">
-<tr><td width="130px" align=center>MilliQ</td><td width="90px"  align=right>6.5 µl</td></tr>
-<tr><td align=center>Flag-tag dMLF</td><td align=right>20 µl</td></tr>
-<tr><td align=center><em>Xba</em>Ⅰ</td><td align=right>0.5 µl</td></tr>
-<tr><td align=center>10 x M Buffer</td><td align=right>3 µl</td></tr>
-<tr><td align=center>&nbsp;</td><td align=right>total 30 µl</td></tr>
-</table>
 <br>
+:Image of agarose gel after DNA fragment isolation.
-(3)<br>
+:<html><body>
-<tr><td><table border=1 width="220px">
+<IMG src="https://static.igem.org/mediawiki/2011/1/1b/2011.09.24_%E3%82%B2%E3%83%AB%E6%8A%BD%E5%87%BA.JPG" width="250px" height="250px" border="0">
-<tr><td width="130px" align=center>MilliQ</td><td width="90px"  align=right>6 µl</td></tr>
+</body></html>
-<tr><td align=center>Flag-tag dMLF</td><td align=right>20 µl</td></tr>
-<tr><td align=center><em>Pst</em>Ⅰ</td><td align=right>0.5 µl</td></tr>
-<tr><td align=center><em>Xba</em>Ⅰ</td><td align=right>0.5 µl</td></tr>
-<tr><td align=center>10 x M Buffer</td><td align=right>3 µl</td></tr>
-<tr><td align=center>&nbsp;</td><td align=right>total 30 µl</td></tr>
-</table>
 <br>
+:A band was seen to about 2kb.
-゜Cで18時間インキュベートした後、アガロースゲル電気泳動を行った。<br>
-<BR>
-ゲル1枚当たりの組成
-<tr><td><table border=1 width="200px">
-<tr><td width="150px" align=center>SeaKem<sup>R</sup>GTG<sup>R</sup>-agar</td><td width="50px" align=right>0.4 g</td></tr>
-<tr><td align=center>1 x TAE</td><td align=right>20 ml</td></tr>
-</table>
-<BR>
-↓上記の組成に従い、試薬を三角フラスコで混ぜてレンジで加熱し、専用容器に入れて固めた<BR>
-↓制限酵素処理した反応液50 µlに対して6 x loading dye を10 µl加えた<BR>
-↓サンプルとDNA maker をコーム穴に入れた<BR>
-↓サンプルをセット後、100 V 20minで電気泳動した<BR>
-↓電気泳動後、EtBrで10minゲルを染色した<BR>
-↓染色後、MilliQでゲルを数回洗ってプレートにのせた<BR>
-↓UVを照射してDNAのバンドを可視化した<BR>
+==''26th,September''==
+<b>Members</b>
 <br>
-<BR>
+:Yoshimura Nakagawa
-【結果】<BR>
+<br>
-泳動後の写真<BR>
+:1.Ligation
-<IMG src="https://static.igem.org/mediawiki/2011/9/98/2011.09.19MLF%E3%83%81%E3%82%A7%E3%83%83%E3%82%AF.JPG
+:2.restriction enzyme handling of liquid pSB1C3
-" width="240px" height="280px" border="0">
-<BR>
-Flag-tag dMLFは<em>Pst</em>Ⅰと<em>Xba</em>Ⅰの制限酵素処理によって切れることはなかった。<BR>
-<BR>
-<BR>
-/19<BR>
-(目的)<BR>
-ベクター、GFPの濃度をもう一度計ってみるために濃度チェックをした<BR>
-ライゲーションの失敗がコンタミしたプレートによるものであると考えて、
-新しいプレートでもう一度GFPとベクターを精製しなおすことにした<BR>
-一応おかしな量ではあるがプレカルチャーをしてみることにした<BR>
-(方法)<BR>
-GFPの濃度チェックをするために以下の希釈を行った<BR>
-<table border=1 width="160px">
-<tr><td width="70px" align=center>1倍希釈</td><td width="90px"  align=right>1µl</td></tr>
-<tr><td align=center>2倍希釈</td><td align=right>1µl</td></tr>
-<tr><td align=center>4倍希釈</td><td align=right>1µl</td></tr>
-<tr><td align=center>8倍希釈</td><td align=right>1µl</td></tr>
-</table><BR>
-GFPの濃度チェックのための電気泳動写真<BR>
-<IMG src="https://static.igem.org/mediawiki/2011/5/52/2011.09.19_GFP%E6%BF%83%E5%BA%A6%E3%83%81%E3%82%A7%E3%83%83%E3%82%AF.JPG" width="250px" height="250px" border="0"><BR>
+:restriction enzymes processing with pSB1C3
+:According to a list shown below, I performed restriction enzyme processing at 37 for 30 minutes.
-(結果)<BR>
+:<table border=1 width="220px">
-GFPの増え方は良好で大きなコロニーの育成が見られた<BR>
+:<tr><td width="130px" align=center>ddH<sub>2</sub>O</td><td width="90px"  align=right>2 µl</td></tr>
-大腸菌ベクターの方もGFP程ではなかったがある程度の数が生えていたのでよかった<BR>
+:<tr><td align=center>BBa_E0240</td><td align=right>50 µl</td></tr>
-一応白く濁りはしていたので大腸菌の増えていることは確認することが出来た<BR>
+:<tr><td align=center><em>Eco</em>RⅠ</td><td align=right>1 µl</td></tr>
-<BR>
+:<tr><td align=center><em>Pst</em>Ⅰ</td><td align=right>1 µl</td></tr>
-/20(火)<BR>
+:<tr><td align=center>10 x H Buffer</td><td align=right>6 µl</td></tr>
-<BR>
+:<tr><td align=center>&nbsp;</td><td align=right>total 60 µl</td></tr>
-アルカリミニプレップ<BR>
+:</table>
-【目的】<BR>
-形質転換した大腸菌からプラスミドDNAを回収、精製する。 <BR>
-<BR>
-【実験方法】<BR>
-<tr><td><table border=1 width="280px">
-<tr><td width="80px" align=center>Solution I</td><td width="200px"  align=right>50 mM グルコース (MW 180)</td></tr>
-<tr><td>&nbsp;</td><td align=right>10 mM EDTA(pH 8.0)</td></tr>
-<tr><td>&nbsp;</td><td align=right>25 mM Tris-HCl (pH 8.0)</td></tr>
-<tr><td align=center>Solution II</td><td align=right>0.2 N NaOH</td></tr>
-<tr><td>&nbsp;</td><td align=right>1% SDS</td></tr>
-<tr><td align=center>Solution III</td><td align=right>3 M 酢酸カリウム</td></tr>
-<tr><td>&nbsp;</td><td align=right>1.8 M 酢酸</td></tr>
-</table><BR>
-<BR>
-↓前日にプレカルチャーした1.5 mlの培養液を1.5 mlチューブにうつした<BR>
-↓15,000 rpm、4°Cで1分間遠心し、上清を捨てた<BR>
-↓100 µlの氷冷したSolution Iを沈殿に加え、懸濁した<BR>
-↓200 µlのSolution IIを加え、混ぜた<BR>
-↓氷上で5分間冷やした<BR>
-↓150 µlの氷冷したSolution IIIを加え、穏やかに反転し混ぜた<BR>
-↓氷上で5分間冷やした<BR>
-↓15,000 rpm、4°Cで5分間遠心した<BR>
-↓400 µlのきれいな上清を注意してピペットで新しいチューブにとった<BR>
-↓900 µlのイソプロパノールを加え、混ぜた<BR>
-↓2分間室温で放置した<BR>
-↓15,000 rpm、4°Cで10分間遠心し、上清を捨てた<BR>
-↓1 mlの70%エタノールを加えた<BR>
-↓ 15,000 rpm、4°Cで2分間遠心し、上清を捨てた<BR>
-↓沈殿を10分～15分乾かした<BR>
-↓プラスミドDNAを30 µlのRNaseのはいったTEに溶かした<BR>
-<BR>
-【結果】<BR>
-<BR>
-<BR>
-トランスフォーメーション<BR>
-【目的】<BR>
-pSB1C3のバックグラウンドチェック<BR>
-<BR>
-【実験方法】<BR>
-↓氷上でコンピテント細胞(XL1-Blue:大腸菌株)を解凍した<BR>
+:Next ligate with following list.
-↓前もって冷やしておいた1.5 mlチューブに100 µlのコンピテント細胞を分注した<BR>
-↓DNAをチューブに1～5 µl加えて、氷上で30分間冷やした<BR>
-↓42°Cで45秒間熱ショックを与えた<BR>
-↓素早く氷上に移し、2分間冷やした<BR>
-↓300 µlのSOC培地を加えた<BR>
-↓37°Cで1時間、振りながら回復培養した<BR>
-↓1 mlをLBプレート(+クロラムフェニコール)にまいた<BR>
-↓37°Cで一晩培養した<BR>
-<BR>
-【結果】<BR>
-<BR>
-<BR>
-ライゲーション<BR>
-【目的】<BR>
-<BR>
-<BR>
-【実験方法】<BR>
-<BR>
-【結果】<BR>
-どのプレートにも無数のコロニーが生えていた。<BR>
-<BR>
-/20<BR>
-(目的)<BR>
-昨日殖やしたおかしなライゲーション産物コロニーのアルカリミニプレップ、MLFの濃度をチェック、ベクターのプレカルチャー<BR>
-(方法)<BR>
-ベクターの方は濃度も薄いということでこれからもどんどん殖やしていこうということにした<BR>
-そこで2mlの培地を8本分殖やしたものをアルカリミニプレップしていった<BR>
-MLFの濃度も昨日同様にニサンプルあるので希釈を四倍までで行っていった<BR>
+:At first I dilute the density of vector to become 10 pg/µl.
-そしてその後の二回目は8倍から32倍の希釈を行ってみた<BR>
+:And I dilute it as I calculate one and MLF 60 ng/µl of GFP 70 ng/µl to be able to go by combination below and am composed follows at 1 µl.
-<tr><td><table border=1 width="160px">
+:I combined it in the ratio of follows afterwards.
-<tr><td width="70px" align=center>MLF</td><td width="90px"  align=right>5μl</td></tr>
-<tr><td align=center>loading dye</td><td align=right>1μl</td></tr>
-</table>
-左　一回目のMLFの電気泳動　右　二回目のMLFの電気泳動<BR>
-<IMG src="https://static.igem.org/mediawiki/2011/a/ac/2011.09.20_MLF%E6%BF%83%E5%BA%A6%E3%83%81%E3%82%A7%E3%83%83%E3%82%AF.JPG
-" width="240px" height="280px" border="0">
-<IMG src="https://static.igem.org/mediawiki/2011/9/9a/2011.09.20_MLF%E6%BF%83%E5%BA%A6%E3%83%81%E3%82%A7%E3%83%83%E3%82%AF%EF%BC%92%E5%9B%9E%E7%9B%AE.JPG
+pSB1C3:GFP＝1:10<BR>
-" width="240px" height="280px" border="0"><BR>
+:<table border=1 width="200px">
+:<tr><td width="130px" align=center>insert</td><td width="70px" align=right>1.0 µl</td></tr>
+:<tr><td align=center>vector</td><td align=right>1.0µl</td></tr>
+:<tr><td align=center>10 x Buffer</td><td align=right>2.5 µl</td></tr>
+:<tr><td align=center>F4 ligase</td><td align=right>0.5 µl</td></tr>
+:<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>1.0 µl</td></tr>
+:<tr><td>&nbsp;</td><td align=right>total 6.0 µl</td></tr>
+:</table>
+pSB1C3:GFP＝1:20<BR>
+:<table border=1 width="200px">
+:<tr><td width="130px" align=center>insert</td><td width="70px" align=right>1.0 µl</td></tr>
+:<tr><td align=center>vector</td><td align=right>1.0 µl</td></tr>
+:<tr><td align=center>10 x Buffer</td><td align=right>2.5 µl</td></tr>
+:<tr><td align=center>F4 ligase</td><td align=right>0.5 µl</td></tr>
+:<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>1.0 µl</td></tr>
+:<tr><td>&nbsp;</td><td align=right>total6.0µl</td></tr>
+:</table>
+pSB1C3:GFP＝1:40<BR>
+:<table border=1 width="200px">
+:<tr><td width="130px" align=center>insert</td><td width="70px" align=right>1.0 µl</td></tr>
+:<tr><td align=center>vector</td><td align=right>1.0 µl</td></tr>
+:<tr><td align=center>10 x Buffer</td><td align=right>2.5 µl</td></tr>
+:<tr><td align=center>F4 ligase</td><td align=right>0.5 µl</td></tr>
+:<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>1.0 µl</td></tr>
+:<tr><td>&nbsp;</td><td align=right>total 6 µl</td></tr>
+:</table>
+:I incubated them in 16°C for 30 min.
+:After that, I transformed them, and I poured them into 300 µl of SOC medium and add them on the plate after culture for 30 minutes.
+<br>
+<b>Results</b>
+:There are no colonies.
-(結果)<BR>
-途中でベクターが消えてしまった<BR>
-MLFのほうは濃度チェックをしてみたところおよそ一回目では濃すぎて分からないので二回目薄くして70ng/µlくらいではないかということが分かりました<BR>
-/21<BR>
-(目的)<BR>
-プレート作成（濃度を分けて）、昨日の大腸菌のアルカリミニプレップ、プレートチェック<BR>
-(方法)<BR>
-プレート自体の劣化も大きく可能性としてあり得るのでここでクローラムフェにコールを新しいものにしの濃度を変化させてプレートを作成した<BR>
-プレート作成の濃度<BR>
-そのⅠ
-<tr><td><table border=1 width="160px">
-<tr><td width="70px" align=center>LB培地</td><td width="90px"  align=right>300ml</td></tr>
-<tr><td align=center>Chloramphenicol</td><td align=right>300μl</td></tr>
-</table>
-そのⅡ
-<tr><td><table border=1 width="160px">
-<tr><td width="70px" align=center>LB培地</td><td width="90px"  align=right>300ml</td></tr>
-<tr><td align=center>Chloramphenicol</td><td align=right>600μl</td></tr>
-</table><tr><td><table border=1 width="160px">
-そのⅢ
-<tr><td width="70px" align=center>LB培地</td><td width="90px"  align=right>300ml</td></tr>
-<tr><td align=center>Chloramphenicol</td><td align=right>1.2ml</td></tr>
-</table>
-<BR>
-上記のプレートを作成し、それぞれ12枚程度できた。<BR>
-もしものために行ったおかしな量であったコロニーを突っついて増えたベクターを精製して電気泳動した。<BR>
+==''29th,September''==
-条件は100v,30分で行った。<BR>
+<b>Members</b>
+<br>
+:Yoshimura,  Nakagawa
+<br>
+:At first I dilute the density of vector to become 10 pg/µl.
+:And I dilute it as I calculate one and MLF 60 ng/µl of GFP 70 ng/µl to be able to go by combination below and am composed follows at 1 µl.
+:I combined it in the ratio of follows afterwards.
-<IMG src="https://static.igem.org/mediawiki/2011/4/41/2011.09.21_%E3%83%99%E3%82%AF%E3%82%BF%E3%83%BC.JPG" width="240px" height="280px" border="0"><BR>
+pSB1C3:GFP＝1:10<BR>
+:<table border=1 width="200px">
+:<tr><td width="130px" align=center>insert</td><td width="70px" align=right>1.0 µl</td></tr>
+:<tr><td align=center>vector</td><td align=right>1.0µl</td></tr>
+:<tr><td align=center>10 x Buffer</td><td align=right>2.5 µl</td></tr>
+:<tr><td align=center>F4 ligase</td><td align=right>0.5 µl</td></tr>
+:<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>1.0 µl</td></tr>
+:<tr><td>&nbsp;</td><td align=right>total 6.0 µl</td></tr>
+:</table>
+pSB1C3:GFP＝1:20<BR>
+:<table border=1 width="200px">
+:<tr><td width="130px" align=center>insert</td><td width="70px" align=right>1.0 µl</td></tr>
+:<tr><td align=center>vector</td><td align=right>1.0 µl</td></tr>
+:<tr><td align=center>10 x Buffer</td><td align=right>2.5 µl</td></tr>
+:<tr><td align=center>F4 ligase</td><td align=right>0.5 µl</td></tr>
+:<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>1.0 µl</td></tr>
+:<tr><td>&nbsp;</td><td align=right>total6.0µl</td></tr>
+:</table>
+pSB1C3:GFP＝1:40<BR>
+:<table border=1 width="200px">
+:<tr><td width="130px" align=center>insert</td><td width="70px" align=right>1.0 µl</td></tr>
+:<tr><td align=center>vector</td><td align=right>1.0 µl</td></tr>
+:<tr><td align=center>10 x Buffer</td><td align=right>2.5 µl</td></tr>
+:<tr><td align=center>F4 ligase</td><td align=right>0.5 µl</td></tr>
+:<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>1.0 µl</td></tr>
+:<tr><td>&nbsp;</td><td align=right>total 6 µl</td></tr>
+:</table>
+:I incubated them in 16°C for 30 min.
+:After that, I transformed them, and I poured them into 300 µl of SOC medium and add them on the plate after culture for 30 minutes.
+<br>
+<b>Results</b>
+:There are some colonies on the plate.
+:However there aren’t any MLF colonies.
-(結果)<BR>
-昨日の大腸菌が多少DNAの沈殿があったので電気泳動したがバンドはできなかった。<BR>
-<BR>
-/22(木)<BR>
-<BR>
-ライゲーション<BR>
-【目的】<BR>
-<BR>
-<BR>
-【実験方法】<BR>
-<BR>
-<BR>
-【結果】<BR>
-<BR>
-<BR>
-/22<BR>
-(目的)<BR>
-ベクターのアルカリミニプレップ、プレカルチャー、プレートチェック<BR>
-(方法)<BR>
-こうなってくると後はくっつくまで試していくしかないのでそのためのベクターの精製とライゲーションを行った<BR>
-最初はベクターのアルカリミニプレップを行った。<BR>
-<BR>
-そして昨日作ったものに今まで使っていたコンピテントセル（XL1-Blue)のみをまいて増えないことを確認してみた<BR>
-その後に今までで成功していたpSB1C3のコロニーでプレカルチャーをした。
-(結果)<BR>
+==''30th,September''==
-プレートチェックは予想通り生えることはなく成功だった、そして形質転換も少なかったがコロニーを確認できた<BR>
+<b>Members</b>
+<br>
+:Yoshimura, Nakagawa
+<br>
+:At first I dilute the density of vector to become 10 pg/µl
+:And I dilute it as I calculate one and MLF 60 ng/µl of GFP 70 ng/µl to be able to go by combination below and am composed follows at 1 µl
+:I combined it in the ratio of follows afterwards
-/23(金)<BR>
+pSB1C3:GFP＝1:10<BR>
-<BR>
+:<table border=1 width="200px">
-トランスフォーメーション<BR>
+:<tr><td width="130px" align=center>insert</td><td width="70px" align=right>1.0 µl</td></tr>
-【目的】<BR>
+:<tr><td align=center>vector</td><td align=right>1.0µl</td></tr>
-<BR>
+:<tr><td align=center>10 x Buffer</td><td align=right>2.5 µl</td></tr>
-<BR>
+:<tr><td align=center>F4 ligase</td><td align=right>0.5 µl</td></tr>
-【実験方法】<BR>
+:<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>1.0 µl</td></tr>
-<BR>
+:<tr><td>&nbsp;</td><td align=right>total 6.0 µl</td></tr>
-<BR>
+:</table>
-【結果】<BR>
+pSB1C3:GFP＝1:20<BR>
-<BR>
+:<table border=1 width="200px">
-<BR>
+:<tr><td width="130px" align=center>insert</td><td width="70px" align=right>1.0 µl</td></tr>
-<BR>
+:<tr><td align=center>vector</td><td align=right>1.0 µl</td></tr>
-<BR>
+:<tr><td align=center>10 x Buffer</td><td align=right>2.5 µl</td></tr>
-<BR>
+:<tr><td align=center>F4 ligase</td><td align=right>0.5 µl</td></tr>
-/23<BR>
+:<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>1.0 µl</td></tr>
-(目的)
+:<tr><td>&nbsp;</td><td align=right>total6.0µl</td></tr>
-ベクターのアルカリミニプレップ、フェノクロ処理、制限酵素処理、ベクターのトランスフォーム、ライゲーション
+:</table>
+pSB1C3:GFP＝1:40<BR>
+:<table border=1 width="200px">
+:<tr><td width="130px" align=center>insert</td><td width="70px" align=right>1.0 µl</td></tr>
+:<tr><td align=center>vector</td><td align=right>1.0 µl</td></tr>
+:<tr><td align=center>10 x Buffer</td><td align=right>2.5 µl</td></tr>
+:<tr><td align=center>F4 ligase</td><td align=right>0.5 µl</td></tr>
+:<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>1.0 µl</td></tr>
+:<tr><td>&nbsp;</td><td align=right>total 6 µl</td></tr>
+:</table>
-(方法)
+:I incubated them in 16°C for 30 min.
-まずくっつかない原因としてやっぱりベクターが悪いのではないかということで新しくプレートにpSB1C3をいれた大腸菌(XL1-Blue)をまいて様子を見てみた<BR>
+:After that, I transformed them, and I poured them into 300 µl of SOC medium and add them on the plate after culture for 30 minutes.
+:After that We did alkaline-lysis method restriction and enzymes processing with ligation products.
+<br>
+<b>Results</b>
+:After all MLF did not exist even if there was the BBa_E0240 in ligation products.
+:And the first turn of parts was completed today.
-そして昨日プレカルチャーしたものをアルカリミニプレした<BR>
-その時二本だけバンドが見つかった。<BR>
-電気泳動写真<BR>
-<IMG src="https://static.igem.org/mediawiki/2011/d/d8/2011.09.23_%E4%B8%AD%E5%B7%9D.JPG" width="240px" height="280px" border="0"><BR>
-ライゲーションを下記のように行った<BR>
- 下記の組成に従って反応液を調整した<BR>
-<BR>
-まず提出用ベクターの濃度を10pg/μlになるように希釈する<BR>
-そして下の配合で行けるようにinsert156ng/μlの方を計算して希釈する<BR>
-その後に下記の割合で配合していった<BR>
-提出用ベクター:GFP＝1:10<BR>
-<table border=1 width="200px">
-<tr><td width="130px" align=center>insert</td><td width="70px" align=right>1.0 µl</td></tr>
-<tr><td align=center>vector</td><td align=right>1.0µl</td></tr>
-<tr><td align=center>10 x Buffer</td><td align=right>2.5 µl</td></tr>
-<tr><td align=center>F4 ligase</td><td align=right>0.5 µl</td></tr>
-<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>1.0 µl</td></tr>
-<tr><td>&nbsp;</td><td align=right>total 6.0 µl</td></tr>
-</table>
-提出用ベクター:GFP＝1:20<BR>
-<table border=1 width="200px">
-<tr><td width="130px" align=center>insert</td><td width="70px" align=right>1.0 µl</td></tr>
-<tr><td align=center>vector</td><td align=right>1.0 µl</td></tr>
-<tr><td align=center>10 x Buffer</td><td align=right>2.5 µl</td></tr>
-<tr><td align=center>F4 ligase</td><td align=right>0.5 µl</td></tr>
-<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>1.0 µl</td></tr>
-<tr><td>&nbsp;</td><td align=right>total6.0µl</td></tr>
-</table>
-提出用ベクター:GFP＝1:40<BR>
-<table border=1 width="200px">
-<tr><td width="130px" align=center>insert</td><td width="70px" align=right>1.0 µl</td></tr>
-<tr><td align=center>vector</td><td align=right>1.0 µl</td></tr>
-<tr><td align=center>10 x Buffer</td><td align=right>2.5 µl</td></tr>
-<tr><td align=center>F4 ligase</td><td align=right>0.5 µl</td></tr>
-<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>1.0 µl</td></tr>
-<tr><td>&nbsp;</td><td align=right>total 6 µl</td></tr>
-</table>
-°C、30minでincubateした
-<BR>
-ライゲーション後、、形質転換を行い、その後にSOC培地300μlに注入し30分培養後プレートにまいた<BR
-(結果)
-たまにバンドは出てくるが全体的に収率が悪いので原因を探ることにする<BR>
-ベクターに問題があるのかもしれないことは大いに考えられるのがライゲーションはこれからも続けていくことになった<BR>
-プレートはきれいだった<BR>
-/24<BR>
-(目的)<BR>
-pSB1C3のアルカリミニプレップ、フェノクロ処理、制限酵素処理、pSB1C3のゲル抽出,GFP制限酵素処理<BR>
-(方法)<BR>
-今回イソプロパノールが切れたので新しいのに代えてみたら格段に収率が上がった<BR>
-これより収率が今まで悪かったのはイソプロパノールの劣化にも一理あると考えられた<BR>
-pSB1C3のアルカリミニプレップ,フェノクロ処理をした後、下表に従って、37℃で一晩制限酵素処理を行った。
-そして以前PCRしたGFPの方も下記に従って制限酵素処理した。<BR>
-<table border=1 width="220px">
-<tr><td width="130px" align=center>ddH<sub>2</sub>O</td><td width="90px"  align=right>2 µl</td></tr>
-<tr><td align=center>BBa_E0240</td><td align=right>50 µl</td></tr>
-<tr><td align=center><em>Eco</em>RⅠ</td><td align=right>1 µl</td></tr>
-<tr><td align=center><em>Pst</em>Ⅰ</td><td align=right>1 µl</td></tr>
-<tr><td align=center>10 x H Buffer</td><td align=right>6 µl</td></tr>
-<tr><td align=center>&nbsp;</td><td align=right>total 60 µl</td></tr>
-</table>
-そして昨日制限酵素処理したpSB1C3をゲル抽出した<BR>
-<a herf="http://www.qiagen.com/products/dnacleanup/gelpcrsicleanupsystems/qiaquickgelextractionkit.aspx">QIAquick Gel Extraction Kit</a>を使用してゲルからpSB1C3のDNAを抽出した。<BR>
-<BR>
-泳動後の写真<BR>
-<IMG src="https://static.igem.org/mediawiki/2011/1/1b/2011.09.24_%E3%82%B2%E3%83%AB%E6%8A%BD%E5%87%BA.JPG" width="250px" height="250px" border="0"><BR>
-約2kbのところにバンドが見られた。
-<BR>
-(結果)
-これでまたライゲーションをの効率を高められるようになった<BR>
-試薬の劣化には注意する必要があることが分かった<BR>
-/25<BR>
-(目的)<BR>
-GFPのPCR,GFPのゲル抽出、GFPの濃度チェック<BR>
-(方法)<BR>
-昨日のGFPをゲル抽出して新たなインサートを作ることにした<BR>
-<a herf="http://www.qiagen.com/products/dnacleanup/gelpcrsicleanupsystems/qiaquickgelextractionkit.aspx">QIAquick Gel Extraction Kit</a>を使用してゲルからpSB1C3のDNAを抽出した。<BR>
-<BR>
-ゲル抽出の写真<BR>
-<IMG src="https://static.igem.org/mediawiki/2011/5/50/2011.09.25_GFP%E3%82%B2%E3%83%AB%E6%8A%BD%E5%87%BA.JPG" width="250px" height="250px" border="0"><BR>
-そして濃度チェックの電気泳動もかけた<BR>
-<table border=1 width="160px">
-<tr><td width="70px" align=center>1倍希釈</td><td width="90px"  align=right>1µl</td></tr>
-<tr><td align=center>2倍希釈</td><td align=right>1µl</td></tr>
-<tr><td align=center>4倍希釈</td><td align=right>1µl</td></tr>
-<tr><td align=center>8倍希釈</td><td align=right>1µl</td></tr>
-</table><BR>
-<IMG src="https://static.igem.org/mediawiki/2011/f/fb/2011.09.25_GFP%E6%BF%83%E5%BA%A6%E3%83%81%E3%82%A7%E3%83%83%E3%82%AF.JPG" width="250px" height="250px" border="0"><BR>
-そして今日もライゲーションをするためにインサートの不足が懸念されたのでPCRもかけた<BR>
-PCR
-<table border="0"><tr><td>
-<table border="0" width="150px">
-<tr><td align=center>PCR条件</td></tr>
-</table>
-<table border=1 width="220px">
-<tr><td width="150px" align=center>10 µM Primer F</td><td width="70px"  align=right>1.5 µl</td></tr>
-<tr><td align=center>10 µM Primer R</td><td align=right>1.5 µl</td></tr>
-<tr><td align=center>GFP</td><td align=right>1 µl</td></tr>
-<tr><td align=center>10 x PCR Buffer for KOD Plus</td><td align=right>5 µl</td></tr>
-<tr><td align=center>dNTPs</td><td align=right>4 µl</td></tr>
-<tr><td align=center>MgSO<sub>4</sub></td><td align=right>4 µl</td></tr>
-<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>32 µl</td></tr>
-<tr><td align=center>KOD Plus</td><td align=right>1 µl</td></tr>
-<tr><td>&nbsp;</td><td align=right>total 50 µl</td></tr>
-</table>
-</td><TD></TD><TD></TD><td>
-<table border="0" width="100px">
-<tr><td align=center>Cycle条件</td></tr>
-</table>
-<table border=1 width="400px">
-<tr><td width="100px" align=center>Pre-Denature</td><td width="100px"  align=center>95°C</td><td width="100px"  align=center>30sec</td></td><td width="100px">&nbsp;</td></tr>
-<tr><td align=center>Denature</td><td align=center>95°C</td><td align=center>30sec</td><td align=center rowspan=3>30 Cycle</td></tr>
-<tr><td align=center>Anneling</td><td align=center>48.5°C</td><td align=center>1min</td></tr>
-<tr><td align=center>Extension</td><td align=center>68°C</td><td align=center>1kb/min</td></tr>
-<tr><td align=center>End</td><td align=center>4°C</td><td align=center>keep</td><td width="100px">&nbsp;</td></tr>
-</table>
-</td></tr>
-</table>
-<BR>
-上記の組成に従って混ぜた<BR>
-GFPのチェックは時間もないのですべて同じ条件より一つを選択して念のためにPCRチェックした<BR>
-PCR増幅チェック<BR>
-<IMG src="https://static.igem.org/mediawiki/2011/1/1e/2011.09.25_GFP-PCR.JPG" width="250px" height="250px" border="0"><BR>
-(結果)<BR>
-GFPは増えていることが分かりゲル抽出からも70ng/μl程度のインサートが取れた<BR>
-/26
-<BR>
-(目的)<BR>
-ライゲーション、液体pSB1C3の制限酵素処理
-(方法)<BR>
-目的のものは十分にそろっているはずなのであとはライゲーションのみに力を入れた<BR>
-また今日から液体状のpSB1C3も制限酵素処理し明日からライゲーションに活用することにした<BR>
-制限酵素処理<BR>
-下表に従って、30分37℃で制限酵素処理を行った。
-<table border=1 width="220px">
-<tr><td width="130px" align=center>ddH<sub>2</sub>O</td><td width="90px"  align=right>2 µl</td></tr>
-<tr><td align=center>BBa_E0240</td><td align=right>50 µl</td></tr>
-<tr><td align=center><em>Eco</em>RⅠ</td><td align=right>1 µl</td></tr>
-<tr><td align=center><em>Pst</em>Ⅰ</td><td align=right>1 µl</td></tr>
-<tr><td align=center>10 x H Buffer</td><td align=right>6 µl</td></tr>
-<tr><td align=center>&nbsp;</td><td align=right>total 60 µl</td></tr>
-</table>
-次にライゲーションを下記のように行った<BR>
- 下記の組成に従って反応液を調整した<BR>
-<BR>
-まず提出用ベクターの濃度を10pg/μlになるように希釈する<BR>
-そして下の配合で行けるようにGFP70ng/μlの方とMLF60ng/μlを計算して1μlで下記の組成になるように希釈する<BR>
-その後に下記の割合で配合していった<BR>
-提出用ベクター:GFP＝1:10<BR>
-<table border=1 width="200px">
-<tr><td width="130px" align=center>insert</td><td width="70px" align=right>1.0 µl</td></tr>
-<tr><td align=center>vector</td><td align=right>1.0µl</td></tr>
-<tr><td align=center>10 x Buffer</td><td align=right>2.5 µl</td></tr>
-<tr><td align=center>F4 ligase</td><td align=right>0.5 µl</td></tr>
-<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>1.0 µl</td></tr>
-<tr><td>&nbsp;</td><td align=right>total 6.0 µl</td></tr>
-</table>
-提出用ベクター:GFP＝1:20<BR>
-<table border=1 width="200px">
-<tr><td width="130px" align=center>insert</td><td width="70px" align=right>1.0 µl</td></tr>
-<tr><td align=center>vector</td><td align=right>1.0 µl</td></tr>
-<tr><td align=center>10 x Buffer</td><td align=right>2.5 µl</td></tr>
-<tr><td align=center>F4 ligase</td><td align=right>0.5 µl</td></tr>
-<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>1.0 µl</td></tr>
-<tr><td>&nbsp;</td><td align=right>total6.0µl</td></tr>
-</table>
-提出用ベクター:GFP＝1:40<BR>
-<table border=1 width="200px">
-<tr><td width="130px" align=center>insert</td><td width="70px" align=right>1.0 µl</td></tr>
-<tr><td align=center>vector</td><td align=right>1.0 µl</td></tr>
-<tr><td align=center>10 x Buffer</td><td align=right>2.5 µl</td></tr>
-<tr><td align=center>F4 ligase</td><td align=right>0.5 µl</td></tr>
-<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>1.0 µl</td></tr>
-<tr><td>&nbsp;</td><td align=right>total 6 µl</td></tr>
-</table>
-°C、30minでincubateした
-<BR>
-ライゲーション後、、形質転換を行い、その後にSOC培地300μlに注入し30分培養後プレートにまいた<BR>
-(結果)<BR>
-やっぱり生えてこなかった<BR>
-おそらくベクターに問題があると考えられる<BR>
-/27<BR>
-(目的)<BR>
-新しいベクターでライゲーション
-(方法)<BR>
-京大さんに頼んで使っている中で余っているpSB1C3を譲っていただきそれでもライゲーションをした<BR>
-<BR>
-ライゲーションを下記のように行った<BR>
- 下記の組成に従って反応液を調整した<BR>
-<BR>
-まず提出用ベクターの濃度を10pg/μlになるように希釈する<BR>
-そして下の配合で行けるようにGFP70ng/μlの方とMLF60ng/μlを計算して1μlで下記の組成になるように希釈する<BR>
-その後に下記の割合で配合していった<BR>
-提出用ベクター:GFP＝1:10<BR>
-<table border=1 width="200px">
-<tr><td width="130px" align=center>insert</td><td width="70px" align=right>1.0 µl</td></tr>
-<tr><td align=center>vector</td><td align=right>1.0µl</td></tr>
-<tr><td align=center>10 x Buffer</td><td align=right>2.5 µl</td></tr>
-<tr><td align=center>F4 ligase</td><td align=right>0.5 µl</td></tr>
-<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>1.0 µl</td></tr>
-<tr><td>&nbsp;</td><td align=right>total 6.0 µl</td></tr>
-</table>
-提出用ベクター:GFP＝1:20<BR>
-<table border=1 width="200px">
-<tr><td width="130px" align=center>insert</td><td width="70px" align=right>1.0 µl</td></tr>
-<tr><td align=center>vector</td><td align=right>1.0 µl</td></tr>
-<tr><td align=center>10 x Buffer</td><td align=right>2.5 µl</td></tr>
-<tr><td align=center>F4 ligase</td><td align=right>0.5 µl</td></tr>
-<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>1.0 µl</td></tr>
-<tr><td>&nbsp;</td><td align=right>total6.0µl</td></tr>
-</table>
-提出用ベクター:GFP＝1:40<BR>
-<table border=1 width="200px">
-<tr><td width="130px" align=center>insert</td><td width="70px" align=right>1.0 µl</td></tr>
-<tr><td align=center>vector</td><td align=right>1.0 µl</td></tr>
-<tr><td align=center>10 x Buffer</td><td align=right>2.5 µl</td></tr>
-<tr><td align=center>F4 ligase</td><td align=right>0.5 µl</td></tr>
-<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>1.0 µl</td></tr>
-<tr><td>&nbsp;</td><td align=right>total 6 µl</td></tr>
-</table>
-°C、30minでincubateした
-<BR>
-ライゲーション後、、形質転換を行い、その後にSOC培地300μlに注入し30分培養後プレートにまいた<BR>
-(結果)<BR>
-今回は液体からのPSB1C3は良くなかったが京大からのはコロニーがあったので大いに期待できた<BR>
-ただMLFはコロニーらしきものもなく大いに心配する出来事だった<BR>
-/28<BR>
-(目的)<BR>
-ライゲーション,ライゲーション産物のプレカルチャー<BR>
-(方法)<BR>
-ライゲーションを下記のように行った<BR>
- 下記の組成に従って反応液を調整した<BR>
-<BR>
-まず提出用ベクターの濃度を10pg/μlになるように希釈する<BR>
-そして下の配合で行けるようにGFP70ng/μlの方とMLF60ng/μlを計算して1μlで下記の組成になるように希釈する<BR>
-その後に下記の割合で配合していった<BR>
-提出用ベクター:GFP＝1:10<BR>
-<table border=1 width="200px">
-<tr><td width="130px" align=center>insert</td><td width="70px" align=right>1.0 µl</td></tr>
-<tr><td align=center>vector</td><td align=right>1.0µl</td></tr>
-<tr><td align=center>10 x Buffer</td><td align=right>2.5 µl</td></tr>
-<tr><td align=center>F4 ligase</td><td align=right>0.5 µl</td></tr>
-<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>1.0 µl</td></tr>
-<tr><td>&nbsp;</td><td align=right>total 6.0 µl</td></tr>
-</table>
-提出用ベクター:GFP＝1:20<BR>
-<table border=1 width="200px">
-<tr><td width="130px" align=center>insert</td><td width="70px" align=right>1.0 µl</td></tr>
-<tr><td align=center>vector</td><td align=right>1.0 µl</td></tr>
-<tr><td align=center>10 x Buffer</td><td align=right>2.5 µl</td></tr>
-<tr><td align=center>F4 ligase</td><td align=right>0.5 µl</td></tr>
-<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>1.0 µl</td></tr>
-<tr><td>&nbsp;</td><td align=right>total6.0µl</td></tr>
-</table>
-提出用ベクター:GFP＝1:40<BR>
-<table border=1 width="200px">
-<tr><td width="130px" align=center>insert</td><td width="70px" align=right>1.0 µl</td></tr>
-<tr><td align=center>vector</td><td align=right>1.0 µl</td></tr>
-<tr><td align=center>10 x Buffer</td><td align=right>2.5 µl</td></tr>
-<tr><td align=center>F4 ligase</td><td align=right>0.5 µl</td></tr>
-<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>1.0 µl</td></tr>
-<tr><td>&nbsp;</td><td align=right>total 6 µl</td></tr>
-</table>
-°C、30minでincubateした
-<BR>
-ライゲーション後、、形質転換を行い、その後にSOC培地300μlに注入し30分培養後プレートにまいた<BR>
-そしてできていたコロニーを培養すべくプレカルチャーをした<BR>
-(結果)<BR>
-京大さんに頂いたものとライゲーションしたGFPのものにまたコロニーがあった。<BR>
-そしてプレカルチャーしていたものも順調に増えていた<BR>
-/29<BR>
-(目的)<BR>
-ライゲーション産物のアルカリミニプレップ、一部を制限酵素処理、ライゲーション、
-(方法)<BR>
-ライゲーション産物をアルカリミニプレップした<BR>
-そして一部をとって電気泳動することにした<BR>
-ライゲーションもまた同様ににした<BR>
-下記の組成に従って反応液を調整した<BR>
-<BR>
-まず提出用ベクターの濃度を10pg/μlになるように希釈する<BR>
-そして下の配合で行けるようにGFP70ng/μlの方とMLF60ng/μlを計算して1μlで下記の組成になるように希釈する<BR>
-その後に下記の割合で配合していった<BR>
-提出用ベクター:GFP＝1:10<BR>
-<table border=1 width="200px">
-<tr><td width="130px" align=center>insert</td><td width="70px" align=right>1.0 µl</td></tr>
-<tr><td align=center>vector</td><td align=right>1.0µl</td></tr>
-<tr><td align=center>10 x Buffer</td><td align=right>2.5 µl</td></tr>
-<tr><td align=center>F4 ligase</td><td align=right>0.5 µl</td></tr>
-<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>1.0 µl</td></tr>
-<tr><td>&nbsp;</td><td align=right>total 6.0 µl</td></tr>
-</table>
-提出用ベクター:GFP＝1:20<BR>
-<table border=1 width="200px">
-<tr><td width="130px" align=center>insert</td><td width="70px" align=right>1.0 µl</td></tr>
-<tr><td align=center>vector</td><td align=right>1.0 µl</td></tr>
-<tr><td align=center>10 x Buffer</td><td align=right>2.5 µl</td></tr>
-<tr><td align=center>F4 ligase</td><td align=right>0.5 µl</td></tr>
-<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>1.0 µl</td></tr>
-<tr><td>&nbsp;</td><td align=right>total6.0µl</td></tr>
-</table>
-提出用ベクター:GFP＝1:40<BR>
-<table border=1 width="200px">
-<tr><td width="130px" align=center>insert</td><td width="70px" align=right>1.0 µl</td></tr>
-<tr><td align=center>vector</td><td align=right>1.0 µl</td></tr>
-<tr><td align=center>10 x Buffer</td><td align=right>2.5 µl</td></tr>
-<tr><td align=center>F4 ligase</td><td align=right>0.5 µl</td></tr>
-<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>1.0 µl</td></tr>
-<tr><td>&nbsp;</td><td align=right>total 6 µl</td></tr>
-</table>
-°C、30minでincubateした
-<BR>
-ライゲーション後、、形質転換を行い、その後にSOC培地300μlに注入し30分培養後プレートにまいた<BR>
-(結果)<BR>
-やはりライゲーションにおいてGFPはあってもMLFは存在しなかった<BR>
-そしてパーツの一つ目は本日完成した<BR>
+<br>
-</p>
-</body>
-</html>