Team:HokkaidoU Japan/Notebook/October2

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===2 piece ligation of T3SS signal and pSB1C3 for DNA Submission===
===2 piece ligation of T3SS signal and pSB1C3 for DNA Submission===
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'''T3SS signal'''
'''T3SS signal'''
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{|style="text-align:center;" class="protocol"

Revision as of 16:29, 2 October 2010

Notebook

Colony PCR

HokkaidoU Japan 20101002a.JPG
  • 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

HokkaidoU Japan 20101002b.JPG

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

HokkaidoU Japan 20101002c.JPG

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

HokkaidoU Japan 20101002d.JPG
HokkaidoU Japan 20101002e.JPG

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===
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