Team:KIT-Kyoto/Notebook/LabNote/GFPMLF8

From 2011.igem.org

(Difference between revisions)
 
(3 intermediate revisions not shown)
Line 15: Line 15:
:Yoshimura, Nakagawa, Matsunami
:Yoshimura, Nakagawa, Matsunami
<br>
<br>
-
:We have transformed E. coli DH5 alpha with the plasmid BBa_E0240.
+
:We have transformed ''E. coli DH5 alpha'' with the plasmid BBa_E0240.
<br>
<br>
<b>Results</b>
<b>Results</b>
Line 21: Line 21:
<br>
<br>
-
==''22rd, August''==
+
==''23rd, August''==
<b>Members</b>
<b>Members</b>
<br>
<br>
Line 77: Line 77:
:<table border="0"><tr><td>
:<table border="0"><tr><td>
:<table border="0" width="150px">
:<table border="0" width="150px">
-
:<tr><td align=center>PCR条件</td></tr>
+
:<tr><td align=center>PCR reaction</td></tr>
:</table>
:</table>
:<table border=1 width="250px">
:<table border=1 width="250px">
Line 125: Line 125:
<br>
<br>
-
==''8月26日''==
+
==''26th, August''==
<b>Member</b>
<b>Member</b>
<br>
<br>
-
:中川<br>
+
:Nakagawa<br>
<br>
<br>
-
:1.ゲルからのDNA抽出
+
:1.Extraction of DNA from the agarose gel.
-
:2.pSB1C3のトランスフォーメーション
+
:2.Transformation of ''E. coli DH5 alpha'' with the plasmid pSB1C3.
<br>
<br>
<b>Results</b>
<b>Results</b>
-
:1.ゲル切り出し後の写真<BR>
+
:1. Picture of agarose gel after cutting out of the portion containing the DNA fragment.<BR>
:<html><body>
:<html><body>
<IMG src="https://static.igem.org/mediawiki/2011/c/ce/8.26GFP%E3%82%B2%E3%83%AB%E6%8A%BD.jpg" width="250px" height="250px" border="0">
<IMG src="https://static.igem.org/mediawiki/2011/c/ce/8.26GFP%E3%82%B2%E3%83%AB%E6%8A%BD.jpg" width="250px" height="250px" border="0">
</body></html>
</body></html>
<br>
<br>
-
:濃度は510 ng/µlだった。
+
:Final concentration of the isolated DNA was 510 ng/µl.
<br>
<br>
-
:2.小さいが、コロニーが生えていた。念のために、翌日再度トランスフォーメーションをする。
+
:2. Although small colonies were observed, we decided to transform the bacteria again just in case.
<br>
<br>
-
==''8月27日''==
+
==''27th, August''==
<b>Member</b>
<b>Member</b>
<br>
<br>
-
:吉村
+
:Yoshimura
<br>
<br>
-
:pSB1C3のトランスフォーメーション
+
:Transformation of ''E. coli DH5'' alpha with the plasmid pSB1C3
<br>
<br>
<b>Results</b>
<b>Results</b>
-
:小さいコロニーの生育がみられた。
+
:The transformed bacterial colonies were obtained.
<br>
<br>
-
==''8月29日''==
+
==''29th, August''==
<b>Member</b>
<b>Member</b>
<br>
<br>
-
:吉村、中川
+
:Yoshimura, Nakagawa
<br>
<br>
-
:pSB1C3のプレカルチャー(6サンプル)
+
:Pre-culture of pSB1C3(6 sample)
<br>
<br>
<b>Results</b>
<b>Results</b>
-
:6サンプルのうち3サンプルは培養に成功した。
+
:We have succeeded to grow bacteria from three samples out of six.
-
:3サンプルはコンタミの可能性があるため、念を入れて翌日もプレカルチャーを行うことにした。
+
<br>
<br>
-
==''8月30日''==
+
==''30th, August''==
<b>Member</b>
<b>Member</b>
<br>
<br>
-
:中川<br>
+
:Nakagawa<br>
<br>
<br>
-
:1.pSB1C3のプレカルチャー(6サンプル)
+
:1.Pre-culture of pSB1C3(6 sample)
-
:2.プライマーの再設計
+
:2.Design of primers to construct GFP plasmid without ATG from the plasmid BBa_E0240.
<br>
<br>
<b>Results</b>
<b>Results</b>
-
:1.コンタミすることなく培養に成功した。
+
:1.We have succeeded the preculture.
-
:2.以下のように設計した。
+
:2.The designed primers are shown below.
::F: 3' TT<span style="COLOR: red">GAATTC</span>TTT<span style="COLOR: red">TCTAGA</span>TTTCGTAAAGGAGAAGAA<br>
::F: 3' TT<span style="COLOR: red">GAATTC</span>TTT<span style="COLOR: red">TCTAGA</span>TTTCGTAAAGGAGAAGAA<br>
            <em>Eco</em>RⅠ     <em>Xba</em>Ⅰ<br>
            <em>Eco</em>RⅠ     <em>Xba</em>Ⅰ<br>
-
::Tm値:68.8゜C  35塩基<br>
+
::Tm value :68.8゜C  35 base<br>
<br>
<br>
-
==''8月31日''==
+
==''31st, August''==
<b>Member</b>
<b>Member</b>
<br>
<br>
-
:中川<br>
+
:Nakagawa<br>
<br>
<br>
-
:pSB1C3のアルカリミニプレップ、フェノールクロロホルム処理をした後、下表に従って、30分37℃で制限酵素処理を行った。
+
:After isolation of pSB1C3 by the alkaline-lysis methodfollowed by phenol-chloroform treatment, DNA was digested with the following restriction enzymes (30 min at 37℃).
:<table border=1 width="220px">
:<table border=1 width="220px">
Line 199: Line 198:
:</table>
:</table>
<br>
<br>
-
:制限酵素処理後、100 V 30minで電気泳動を行った。
+
:After restriction enzyme digestion, DNA samples were electrophoresed at 100 V for 30min.
<br>
<br>
<b>Results</b>
<b>Results</b>
-
:泳動後の写真<BR>
+
:Image of the agarose gel.<BR>
:<html><body>
:<html><body>
<IMG src="https://static.igem.org/mediawiki/2011/f/f2/2011.08.31.JPG" width="250px" height="250px" border="0">
<IMG src="https://static.igem.org/mediawiki/2011/f/f2/2011.08.31.JPG" width="250px" height="250px" border="0">

Latest revision as of 03:40, 6 October 2011



Home Team Project Parts Notebook Safety Human Practice Attributions


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

22nd, August

Members

Yoshimura, Nakagawa, Matsunami


We have transformed E. coli DH5 alpha with the plasmid BBa_E0240.


Results

The transformed bacterial colonies were obtained.


23rd, August

Members

Yoshimura, Nakagawa


1. Pre-culture of bacteria transformed with BBa_E0240.
2. Design of primers to construct GFP plasmid without ATG from the plasmid BBa_E0240.


Results

1. We have succeeded the preculture.
2. The designed primers are shown below.
F: 3' TTGAATTCTTTCTAGATTCGTAAAGGAGAAGAA

         EcoRⅠ   Xba

Tmvalue:67.75°C  33 base


R: 5' AAACTGCAGAAAACTAGTTTTTTATTATTTGTATAG

           PstⅠ   Spe

Tmvalue:67.66°C  36 base


24th, August

Members

Yoshimura、Nakagawa


After isolation of plasmid DNA BBa_E0240 by the alkaline-lysis method, DNA samples were digested by the following restriction enzymes at 37 ℃ for 30 min.
ddH2O2 µl
BBa_E024050 µl
EcoRⅠ1 µl
Pst1 µl
10 x H Buffer6 µl
 total 60 µl


After restriction enzyme digestion, samples were electrophoresed in 1% agarose gel at 100 V for 30min.


Results

DNA bands in the agarose gel.


You can see DNA band at around 800bp.


25th, August

Members

Yoshimura、Nakagawa


PCR reaction was carried out by using primers designed on 23rd. The reaction conditions are summarized below.
PCR reaction
10 µM GFP Primer F1.5 µl
10 µM GFP Primer R1.5 µl
BBa_E02401 µl
10 x PCR Buffer for KOD Plus5 µl
dNTPs4 µl
MgSO44 µl
ddH2O32 µl
KOD Plus1 µl
 total 50 µl
Cycle条件
Pre-Denature95°C30sec 
Denature95°C30sec30 Cycle
Anneling50°C1min
Extension68°C1min
End4°Ckeep 
Agarose gel electrophoresis was carried out to detect the PCR products.
Then the PCR products were digested with the following restriction enzymes (at 37°C for 16 hours).
ddH2O2 µl
BBa_E024050 µl
EcoRⅠ1 µl
Pst1 µl
10 x H Buffer6 µl
 total 60 µl


Results

DNA bands in the agarose gel.


You can see DNA band at around 800bp.


26th, August

Member

Nakagawa


1.Extraction of DNA from the agarose gel.
2.Transformation of E. coli DH5 alpha with the plasmid pSB1C3.


Results

1. Picture of agarose gel after cutting out of the portion containing the DNA fragment.


Final concentration of the isolated DNA was 510 ng/µl.


2. Although small colonies were observed, we decided to transform the bacteria again just in case.


27th, August

Member

Yoshimura


Transformation of E. coli DH5 alpha with the plasmid pSB1C3


Results

The transformed bacterial colonies were obtained.


29th, August

Member

Yoshimura, Nakagawa


Pre-culture of pSB1C3(6 sample)


Results

We have succeeded to grow bacteria from three samples out of six.


30th, August

Member

Nakagawa


1.Pre-culture of pSB1C3(6 sample)
2.Design of primers to construct GFP plasmid without ATG from the plasmid BBa_E0240.


Results

1.We have succeeded the preculture.
2.The designed primers are shown below.
F: 3' TTGAATTCTTTTCTAGATTTCGTAAAGGAGAAGAA

         EcoRⅠ    Xba

Tm value :68.8゜C  35 base


31st, August

Member

Nakagawa


After isolation of pSB1C3 by the alkaline-lysis methodfollowed by phenol-chloroform treatment, DNA was digested with the following restriction enzymes (30 min at 37℃).
ddH2O2 µl
BBa_E024050 µl
EcoRⅠ1 µl
Pst1 µl
10 x H Buffer6 µl
 total 60 µl


After restriction enzyme digestion, DNA samples were electrophoresed at 100 V for 30min.


Results

Image of the agarose gel.