Team:HokkaidoU Japan/Notebook/October2
From 2010.igem.org
(Difference between revisions)
(→2 piece ligation of T3SS signal and pSB1C3 for DNA Submission) |
|||
Line 134: | Line 134: | ||
|} | |} | ||
</div> | </div> | ||
+ | <div style="clear:both;"></div> | ||
->Incubated at 37C for 2 hrs | ->Incubated at 37C for 2 hrs | ||
* Electrophoresis (From left to right:λ/''Hin''dIII 2uL, T3SS signal, pSB1C3, T3SS signal for 3 piece ligation(applied to 2 wells)) | * Electrophoresis (From left to right:λ/''Hin''dIII 2uL, T3SS signal, pSB1C3, T3SS signal for 3 piece ligation(applied to 2 wells)) |
Revision as of 16:29, 2 October 2010
Colony PCR
- Performed Colonie PCR for 3 colonies which were incubated over night
- Colony numbers were: 1, 2 and 3
- Results showed no insertion
- but when result came, had already done miniprep and prepared Sequencing Master Mix
Miniprep
- Used Qiagen kit
- Melted in 50 uL TE instead of H2O
Preparation for Sequencing
- Mixed as shown in the table below
5x Sequencing Buffer 1.5uL 24.75 uL Ready Reaction Premix 1 uL 16.5 H2O 5 uL 80 uL total 7.5/sample
Reagent | Amount | Amount for 16.5 |
---|---|---|
5x Sequencing Buffer | 1.5uL | 24.75 uL |
Ready Reaction Premix | 1 uL | 16.5 |
H2O | 5 uL | 80 uL |
Total | 7.5/sample | 121.25 |
3 Piece Ligation: Retry
Gel Extraction
10月1日に制限酵素処理したDNA solutionをゲル抽
- T3SS signalはバンドが見えなかったため,制限酵素処理からやり直し.原因不明.
Digestion
Reagent | Amount |
---|---|
10x M buffer | 10 uL |
DW | 64 |
T3SS signal | 6 |
BSA | 10 |
Xba I | 9.6 |
Pst I | 0.4 |
Total | 100 uL |
2 piece ligation of T3SS signal and pSB1C3 for DNA Submission
T3SS signal
Reagent | Amount |
---|---|
10x M buffer | 2 uL |
DW | 12 |
DNA | 3 |
BSA | 2 |
EcoR I | 0.5 |
Pst I | 0.5 |
Total | 20 uL |
pSB1C3
Reagent | Amount |
---|---|
10x M buffer | 2 uL |
DW | 13 |
DNA | 2 |
BSA | 2 |
EcoR I | 0.5 |
Pst I | 0.5 |
Total | 20 uL |
->Incubated at 37C for 2 hrs
- Electrophoresis (From left to right:λ/HindIII 2uL, T3SS signal, pSB1C3, T3SS signal for 3 piece ligation(applied to 2 wells))
Colony PCR
遅れてコロニーが出現したため,これらのうち12個(No.5~16)をPCR
- 8, 10, 11, 12, 15が当たりっぽい.
- 13, 14は微妙?
- 8, 10, 11, 12, 13, 14, 15, 16を2 mLのLBT(Tetracycline 15 ug/mL)に移して,培養
- 16はコントロール
- 翌日,Miniprepして一部をNot Iで処理,一部をシークエンサーにかける
- Not Iで処理すると, およそ1800 bpのインサートが現れるはず
T3SSのコロP
送られてきたBAC cloneからコロP
- Colony solution 7 uL, quick Taq 10 uL, primer(Forward and Reverse) 1.5 uL each
- Extention: 90 sec, 40 cycles
->電気泳動,ゲル抽
- 濃度推定(λ/HindIII 2 uL, DNA solution 1 uL)
->40 ng/uL
===Digestion===