User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/06/16

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Bench work

  1. Glycerol stocks
    • 750μL pTXB1His/DH10B QC colony #2 culture from yesterday + 250μL 60% sterile glycerol → tube #70
    • 750μL pTXB1His/DH10B QC colony #3 culture from yesterday + 250μL 60% sterile glycerol → tube #71
    • 750μL pTXB1/DH10B colony #1 culture from yesterday + 250μL 60% sterile glycerol → tube #72
    • → -80°C
  2. Miniprep O/N cultures from yesterday
  3. Quantify [minipreps]
    1. 95μL water + 5μL DNA from miniprep
      • → read A260 and A280
  4. NheI digest
    • performing sequential double digest, so use optimal buffer for SapI instead of NEB buffer 2
    • modify NheI digest protocol as follows
Tube sterile H2O 10X NEB4 100X BSA DNA NheI (10 kU/mL)
pTXB1His#1 34.5 μL 5 μL 0.5 μL 5 μL of 102 μg/mL 5μL
pTXB1 34.5 μL 5 μL 0.5 μL 5 μL of 82 μg/mL 5μL
BSA PCR 24.5 μL 5 μL 0.5 μL 15 μL directly from PCR reaction 5μL
  • → 2h @ 37°C
  • → 20' @ 65°C (it took awhile to get the block to the right temp, so they were on ice for a bit, then at ~70°C before the block reached 65°C)
  • Note: I should have done a PCR cleanup on the BSA PCR sample! I will repeat this. *Kathryn Muratore 10:20, 20 June 2011 (EDT):
    • In retrospect, I'm not sure that I even used the correct tube here *Kathryn Muratore 16:26, 20 June 2011 (EDT):
      • I did use the right tube, as seen on the gels later that week *Kathryn Muratore 10:33, 28 June 2011 (EDT):
  • → store @ -20°C

Results

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