Team:KIT-Kyoto/Notebook/LabNote/GFPMLF9
From 2011.igem.org
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:<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>0.019</td></tr> | ||
- | :<tr><td align=center>2</td><td align=right> | + | :<tr><td align=center>2</td><td align=right>13.0</td></tr> |
- | :<tr><td align=center>3</td><td align=right>0 | + | :<tr><td align=center>3</td><td align=right>19.0</td></tr> |
- | :<tr><td align=center>4</td><td align=right>0 | + | :<tr><td align=center>4</td><td align=right>22.0</td></tr> |
- | :<tr><td align=center>5</td><td align=right>0 | + | :<tr><td align=center>5</td><td align=right>16.0</td></tr> |
- | :<tr><td align=center>ave.</td><td align=right> | + | :<tr><td align=center>ave.</td><td align=right>17.8</td></tr> |
:</table> | :</table> | ||
Revision as of 01:02, 6 October 2011
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Home > Notebook > Lab Note > September | Language:English/Japanese |
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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 - <t>
KOD Plus 1 µl </tr>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
1 0.019 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条件 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条件 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.
9/13(火)
吉村
アガロースゲル電気泳動
【目的】
PCRで目的のDNAが増幅しているかを調べる。
【実験方法】
【結果】
泳動後の写真
<IMG src="
" width="240px" height="280px" border="0">
PCR条件(1)の方が少しバンドが濃くなっていた。
マーカーが流れてしまった。
次回からPCR条件(1)の条件でPCRを行う。
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。
Flag-tag dMLFの制限酵素処理
【目的】
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
【実験方法】
下表に従って、3サンプル制限酵素処理を行った。
(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 |
37゜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。
【結果】
泳動後の写真
<IMG src="
" width="240px" height="280px" border="0">
<IMG src="
" width="240px" height="280px" border="0">
Flag-tag dMLFはPstⅠとXbaⅠの制限酵素処理によって切れることはなかった。
マーカーが開ききっていなかった。
【考察】
今回の実験ではFlag-tag dMLFはPstⅠとXbaⅠ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。
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。
次回はゲルの濃度を2倍にして電気泳動を行う。
PCR
【目的】
9/6に作製したプライマーを用いてFlag-tag dMLFを増幅させる
【実験方法】
以下の条件で4サンプルPCRを行った。
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