User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/07/26

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Objective

  • Clean-up of BSA PCR product from 7/25
  • Double digest (NheI and SapI) of pTXB1 (miniprep from 6/7 and pTXB1.His (Colony #1 miniprep from 6/15) plasmids

and cleaned-up BSA PCR product from above

  • Re-clean EcoRI and phosphatase digested pTXB1 and cut with BamHI → then clean again and ligate
  • Streak plates w/ pTXB1.His and pTXB1 from glycerol stocks # 69 and #72, respectively.

Bench work

  1. Promega Wizard® DNA clean-up of pTXB1 EcoRI/phosphatase digest samples from 7/25
    • Two samples: one phosphatase(+) and the other phosphatase(-)
  2. Promega Wizard® Gel and PCR DNA clean-up of 7/25 BSA PCR product
    • Two samples: BSA PCR and (-)control
  3. Double-digest protocol on two plasmid samples and one insert sample using NheI and SapI:
    • pTXB1.His miniprep from 6/15
    • pTXB1 miniprep from 6/7
    • Cleaned BSA PCR product from #2 just above
    • The protocol taken from 6/28 but with 100μL reaction volume
    • After digest, on ice for 2 hours then heat inactivated @65°C for 30 minutes
  4. Digest of #1 above (pTXB1 w/ EcoRI cut and phosphatase(+/-))
    • 10μL DNA sample + 29.5μL dH2O + 5μL 10x NEB3 + 0.5μL 100x BSA + 5μL BamHI
    • Digest for 2 hours at 37°C, then cleaned once more
  5. Ligation of BamHI digest from #4 just above
    • 10μL DNA sample + 7μL dH2O + 2μL T4 DNA ligase buffer + 1μL T4 DNA ligase
    • 16°C O/N

Results

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