User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/06/21

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Contents

Bench work

  1. SapI digest
    1. 45μL NheI digested BSA PCR from yesterday + 5μL SapI
    2. 45μL NheI digested pTXB1His #1 from last week + 5μL SapI
    3. 45μL NheI digested pTXB1 from last week + 5μL SapI
      • → 2h @ 37°C
      • → 20' @ 65°C
  2. Analytical minigel
    1. 5μL Hartings PCR
    2. 10μL 1KB ladder
    3. 5μL BSA PCR (purified)
    4. 2.5μL pTXB1His #1
    5. 2.5μL miniprepped pTXB1
    6. 5μL (incorrect) NheI digested BSA PCR from last week
    7. 5μL NheI digested BSA PCR from yesterday
    8. 5μL NheI digested pTXB1His #1 from last week
    9. 5μL NheI digested pTXB1 from last week
    10. 5μL NheI/SapI double-digested BSA PCR from yesterday
    11. 5μL NheI/SapI double-digested pTXB1His #1 from last week
    12. 5μL NheI/SapI double-digested pTXB1 from last week
      • loaded double-digest samples prior after the 37°C incubation, but before the 65°C heat-kill was completed
      • Matt Hartings poured the gel - it should be 1%
      • → 45' @ 90V
      • → stain in EtBr
  3. Phosphatase vector
    1. add 1μL of Muratore:Materials/Antarctic Phosphatase to tubes #2 and #3 from step #1 (1.2 and 1.3 → 3.2 and 3.3)
      • → 15' @ 37°C
  4. Gel purification
    • 1.2% gels
    • each well only holds about 15μL sample with large comb, so needed two gels to purify all 3 double-digests
      • First gel leaked a little while setting, but I don't think this significantly affected the well volume
      • Taped 3 lanes together for second gel, and this held all 60μL (50μL rxn + 10μL loading buffer)
    1. First gel
      1. 15μL phosphatased NheI/SapI pTXB1His#1 (3.2 above)
      2. 15μL phosphatased NheI/SapI pTXB1His#1 (3.2)
      3. 15μL phosphatased NheI/SapI pTXB1His#1 (3.2)
      4. 15μL phosphatased NheI/SapI pTXB1His#1 (3.2)
      5. 15μL NheI/SapI BSA PCR (from step #1 → 1.1)
      6. 15μL NheI/SapI BSA PCR (1.1)
      7. 15μL NheI/SapI BSA PCR (1.1)
      8. 15μL NheI/SapI BSA PCR (1.1)
    2. Second gel
      1. --
      2. --
      3. --
      4. 10μL 1KB ladder
      5. --
      6. 60μL phosphatased NheI/SapI pTXB1His#1 (3.3 above)
      • run in tandem; 50' @ 90V
      • stain in EtBr
      • cut out samples:
      1. dd Insert (1.1):
        • 1.6759g-1.0282g = 647.7mg
        • add 650μL Membrane Binding Solution
      2. dd Vector(His) (3.2):
        • 1.5500g-1.0275g = 523.5mg
        • add 530μL Membrane Binding Solution
      3. dd Vector (3.3):
        • 1.5304g-1.0320g = 498.4mg
        • add 500μL Membrane Binding Solution
      • follow Wizard SV Gel and PCR Clean-up kit vacuum protocol instructions
        • each sample took 2 passes through column (<700 μL each time)
        • I failed to incubated the dissolved gel solution on column before applying vacuum
      • store @ -20°C

Results

Analytical minigel

  • Ladder apparently leaked into next lane. Otherwise, everything looks perfect.
  • Surprising that the [insert] seems so much greater than [vector]
  • Quantified [DNA] to estimate I:V ratio for ligation *Kathryn Muratore 12:28, 23 June 2011 (EDT):
    • Used ImageJ and the "Integrated Density" data from the "Measure" function
View/Edit Spreadsheet
Kathryn Muratore 13:46, 24 June 2011 (EDT)
  • The mass of each band in the ladder was incorrect, which made my calculations for the ligation reaction incorrect.
Kathryn Muratore 10:22, 28 June 2011 (EDT)
  • Actually, although the calculations were off by 8-fold in terms of mass, the final [DNA] was 3 higher than the upper-bounds of the recommended amount in terms of mass (1-10μg/mL), but at the low end of the recommended amount in terms of moles (0.1-1μM). Fortuitously, I think this was the right amount of DNA to use in this ligation.
  • Kathryn Muratore 10:08, 28 July 2011 (EDT): I did not subtract the background previously. This is now corrected.

First gel purification

Second gel purification


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