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

3 Piece Ligationやり直し

ゲル抽

10月1日に制限酵素処理したDNA solutionをゲル抽

• 左からλ/HindIII, pSB1T3, pSB1A3, Arabinose Promoter(3-20B), T3SS signal, T3SS signal
  • T3SS signalはバンドが見えないため，制限酵素処理からやり直し．原因不明．

Colony PCR