User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/06/22

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Bench work

  1. Ligation
    • Insert = gel-purified double-digested BSA PCR from yesterday
    • Vector and Vector(His) = gel-purified double-digested pTXB1 or pTXB1His, respectively, from yesterday
    1. 5μL Insert + 10μL Vector(His) + 2μL ligase buffer + 2μL H2O + 1μL T4 DNA Ligase
    2. 5μL Insert + 10μL Vector + 2μL ligase buffer + 2μL H2O + 1μL T4 DNA Ligase
      • → 20' @ 37°C
      • → 15' @ 65°C
      • Note that I should have done a control reaction (vector without insert)
      • Kathryn Muratore 13:55, 24 June 2011 (EDT): Also note that I had 8 times more DNA in the reaction than intended (see yesterday's results)
  2. Transform DH10B
    1. Followed standard transformation protocol for each sample in step #1
      • → heat shock step was 1'20" (intended to do 1' flat)
      • → plated 200μL each on LBAmp and stored the rest @ 4°C
        • → 37°C O/N
  3. Streak ER2566
    1. Streaked out cell stock from NEB onto LB
      • → 37°C O/N
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