User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/07/05

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Objective

Add today's objective(s) here...

Bench work

  1. Analytical gel
    1. ladder
    2. --
    3. Matt Hartings' sample
    4. - 6. --
    5. 5μL 1:10 pTXB1His
    6. 2.5μL gel-purified double-digested insert from last week
    7. 2.5μL gel-purified double-digested vector(His) from last week
    8. 2.5μL gel-purified double-digested vector from last week
    9. 5μL V(His)+I ligation from last week
    10. 5μL V+I ligation from last week
  2. → 50' @ 90V
  3. → stain in EtBr
  4. Transformation
    1. 100μL DH10B + 5μL V(His)+I from last week (same as what is on gel)
    2. 100μL DH10B + 5μL V+I from last week (same as what is on gel)
    3. 100μL DH10B + 5μL V(His) neg ctrl from last week
    4. 100μL DH10B + 5μL V neg ctrl from last week
    • heat shock is 45s @ 42°C
    • plate 100μL on LBAmp100
    • → 37°C O/N
  5. Miniprep
    1. 10 mL LB + 10μL Amp100 + V+I in DH10B colony from last week's transformation (100μL plate)
    2. 10 mL LB + 10μL Amp100 + V+I in NovaBlue colony from last week's transformation, but the 450μL re-plate
    • → 37°C O/N w/ shaking

Results

Image:Dna gel 07052011 pic 2 annotated.jpg

  • can't see undigested vector
  • gel-purified samples did not float away and are visible on gel (good!)
  • clearly see insert and double-inserts (~3kb). Faint band visible at correct size for vector+insert ligation (~7kb) but hard to see in photo.
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