User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/06/28

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Objective

Troubleshoot ligation (see notes from last week)

  • transform old ligation into commercial cells
  • re-ligate O/N @ 16°C
  • start double-digest again

Prep media, if needed, for ER2566 competent cells (just make and use fresh once DNA is ready)

Bench work

  1. Double-digest
Tube sterile H2O 10X NEB2 100X BSA DNA NheI (10 kU/mL) SapI (2 kU/mL
pTXB1His 34.5 μL 5 μL 0.5 μL 10 μL of 102 μg/mL 2.5μL 2.5μL
pTXB1 34.5 μL 5 μL 0.5 μL 10 μL of 82 μg/mL 2.5μL 2.5μL
BSA PCR 24.5 μL 5 μL 0.5 μL 15 μL from purified PCR reaction 2.5μL 2.5μL
  • → 2h @ 37°C
  • → 30' @ 65°C
    • 20' is sufficient
  • → store insert reaction @ -20°C
  1. Phosphatase vectors
    • add 1μL Alkaline Phosphatase to double-digest of pTXB1His and pTXB1 from step #1
    • → 15' @ 37°C
    • → store @ -20°C
  2. Transformation into NovaBlue
    1. 5μL pTXB1His+BSA ligation + 50μL cells
    2. 5μL pTXB1+BSA ligation + 50μL cells
    • Followed standard transformation protocol for each sample in step #1
    • → heat shock step was 45"
    • → plated 100μL each on LBAmp and stored the rest @ 4°C
      • → 37°C O/N
  • Kathryn Muratore 15:49, 29 June 2011 (EDT): I forgot to do the O/N ligation @ 16°C!
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