Bench work
- Ligation
- Insert = gel-purified double-digested BSA PCR from yesterday
- Vector and Vector(His) = gel-purified double-digested pTXB1 or pTXB1His, respectively, from yesterday
- 5μL Insert + 10μL Vector(His) + 2μL ligase buffer + 2μL H2O + 1μL T4 DNA Ligase
- 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)
- Transform DH10B
- 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
- Streak ER2566
- Streaked out cell stock from NEB onto LB
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AU CHEM-570 Lab Prep
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