Team:HokkaidoU Japan/Notebook/October2

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

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===