Bench work
- SapI digest
- 45μL NheI digested BSA PCR from yesterday + 5μL SapI
- 45μL NheI digested pTXB1His #1 from last week + 5μL SapI
- 45μL NheI digested pTXB1 from last week + 5μL SapI
- Analytical minigel
- 5μL Hartings PCR
- 10μL 1KB ladder
- 5μL BSA PCR (purified)
- 2.5μL pTXB1His #1
- 2.5μL miniprepped pTXB1
- 5μL (incorrect) NheI digested BSA PCR from last week
- 5μL NheI digested BSA PCR from yesterday
- 5μL NheI digested pTXB1His #1 from last week
- 5μL NheI digested pTXB1 from last week
- 5μL NheI/SapI double-digested BSA PCR from yesterday
- 5μL NheI/SapI double-digested pTXB1His #1 from last week
- 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
- Phosphatase vector
- 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)
- 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)
- First gel
- 15μL phosphatased NheI/SapI pTXB1His#1 (3.2 above)
- 15μL phosphatased NheI/SapI pTXB1His#1 (3.2)
- 15μL phosphatased NheI/SapI pTXB1His#1 (3.2)
- 15μL phosphatased NheI/SapI pTXB1His#1 (3.2)
- 15μL NheI/SapI BSA PCR (from step #1 → 1.1)
- 15μL NheI/SapI BSA PCR (1.1)
- 15μL NheI/SapI BSA PCR (1.1)
- 15μL NheI/SapI BSA PCR (1.1)
- Second gel
- --
- --
- --
- 10μL 1KB ladder
- --
- 60μL phosphatased NheI/SapI pTXB1His#1 (3.3 above)
- run in tandem; 50' @ 90V
- stain in EtBr
- cut out samples:
- dd Insert (1.1):
- 1.6759g-1.0282g = 647.7mg
- add 650μL Membrane Binding Solution
- dd Vector(His) (3.2):
- 1.5500g-1.0275g = 523.5mg
- add 530μL Membrane Binding Solution
- 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
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- 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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