User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/07/06

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Objective

Improve transformation efficiency

Bench work

  1. Transform NovaBlue
    1. 2.5μL V(His)+I ligation from last week + 25μL NovaBlue
    2. 2.5μL V(His) neg ctrl ligation from last week + 25μL NovaBlue
    • follow transformation protocol
    • heat shock is 80s @ 42°C
    • add 450μL SOC for recovery
    • → plate 100μL on LBAmp100
    • → 37°C O/N
  2. Re-plate transformants
    • spread 450μL of DH10B transformants from yesterday on LBAmp100
    • → 37°C O/N
  3. Electrocompetent DH10B - starter culture
    • 3 mL LB + 50μL E. coli DH10B from thawed chemically-competent cells
    • → 37°C O/N w/ shaking
  4. Culture ER2566
    • for glycerol stock and also to confirm that media is okay since there have been problems with some of my students growing starter cultures.
    • 3 mL LB + colony E. coli ER2566 from streaked plate
    • → 37°C O/N w/ shaking
  5. Test Amp100
    • #* 3 mL LB + DH10B/pKSeAATHis colony #1 from one of Tamra's transformation tests this week x 2
    • → 37°C O/N w/ shaking

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