User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/06/13

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Bench work

  1. Analytical minigel
    • 1.2% agarose gel
    1. --
    2. 10μL 1KB ladder
    3. --
    4. 5μL QuikChange experimental (Dan's sample from 6/10/11)
    5. 5μL QuikChange (-) control (Dan's sample from 6/10/11)
    6. -10 --
      • → ~50' @ 90V
      • → stain with EtBr
  2. DpnI digestion
    • add 1μL DpnI to each of Dan's QuikChange reactions
    • → 1h @ 37°C
    • → store @ -20°C
  3. Transform DH10B
    1. 5μL DpnI-digested QuikChange experimental reaction + 200μL competent DH10B
    2. 5μL DpnI-digested QuikChange control reaction + 200μL competent DH10B

Results

DNA gel 110613 annotated.jpg

  • Perfect! Expected a single band at ~6.7 kb, and that's what we got. For this tricky multiple-base insertion, the 2-stage QuikChange with 5 cycles in the 1st stage is the solution.