Team:KIT-Kyoto/Notebook/LabNote/GFPMLF9
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+ | <b>Results</b> | ||
+ | |||
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+ | ==''8th September''== | ||
+ | <b>Member</b> | ||
+ | <br> | ||
+ | :Nakagawa | ||
+ | <br> | ||
+ | :Extract the DNA of Flag tag dMLF from the gel | ||
+ | |||
+ | ゲル抽出 プロトコル | ||
+ | |||
+ | :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). | ||
+ | |||
+ | :<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> </td><td align=right>total 60 µl</td></tr> | ||
+ | :</table> | ||
+ | |||
+ | <b>Results</b> | ||
+ | :We cannot see any bands of MLF from the gel | ||
+ | :So, we are effort to ligate with pSB1C3 and BBa_E0240 | ||
+ | |||
+ | |||
<BR> | <BR> | ||
<BR> | <BR> |
Revision as of 00:26, 6 October 2011
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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 30rd. 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 0.013 3 0.019 4 0.022 5 0.016 ave. 0.0178
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
Results
8th September
Member
- Nakagawa
- Extract the DNA of Flag tag dMLF from the gel
ゲル抽出 プロトコル
- 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
9/8(木)
中川
(目的)
・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
(方法)
MLFのゲル抽出
<a herf="http://www.qiagen.com/products/dnacleanup/gelpcrsicleanupsystems/qiaquickgelextractionkit.aspx">QIAquick Gel Extraction Kit</a>を使用した
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
そののち制限酵素処理をした
制限酵素処理(提出用ベクター)
下記の組成に従って反応液を調整した
提出用ベクターpSB1C3in ddH2O | 50 µl |
EcoRⅠ | 1 µl |
PstⅠ | 1 µl |
10 x H Buffer | 6 µl |
total 58 µl |
37°Cで20時間静置した
(結果)
MLFを切り出すときにほとんどバンドは見えなかった。
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。
9/9★おk★
(目的)
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
(方法)
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。
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
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
制限酵素処理が完全にし切れていないのではないか
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
(結果)
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。
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
9/12(月)
吉村、中川
PCR
【目的】
9/6に作製したプライマーを用いてをFlag-tag dMLFを増幅させる
【実験方法】
以下の2条件で各1サンプルずつPCRを行った。
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