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

{| width="800"
 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] AU CHEM-570 Lab Prep
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


 * colspan="2"|
 * colspan="2"|

Bench work

 * 1) SapI digest
 * 2) 45μL NheI digested BSA PCR from  yesterday + 5μL SapI
 * 3) 45μL NheI digested pTXB1His #1 from  last week + 5μL SapI
 * 4) 45μL NheI digested pTXB1 from  last week + 5μL SapI
 * 5) *&rarr; 2h @ 37°C
 * 6) *&rarr; 20' @ 65°C
 * 7) Analytical minigel
 * 8) 5μL Hartings PCR
 * 9) 10μL  1KB ladder
 * 10) 5μL  BSA PCR (purified)
 * 11) 2.5μL  pTXB1His #1
 * 12) 2.5μL  miniprepped pTXB1
 * 13) 5μL (incorrect) NheI digested BSA PCR from  last week
 * 14) 5μL NheI digested BSA PCR from  yesterday
 * 15) 5μL NheI digested pTXB1His #1 from  last week
 * 16) 5μL NheI digested pTXB1 from  last week
 * 17) 5μL NheI/SapI double-digested BSA PCR from  yesterday
 * 18) 5μL NheI/SapI double-digested pTXB1His #1 from  last week
 * 19) 5μL NheI/SapI double-digested pTXB1 from  last week
 * 20) * loaded double-digest samples prior after the 37°C incubation, but before the 65°C heat-kill was completed
 * 21) * Matt Hartings poured the gel - it should be 1%
 * 22) *&rarr; 45' @ 90V
 * 23) *&rarr; stain in EtBr
 * 24) Phosphatase vector
 * 25) add 1μL of Muratore:Materials/Antarctic Phosphatase to tubes #2 and #3 from step #1 (1.2 and 1.3 &rarr; 3.2 and 3.3)
 * 26) *&rarr; 15' @ 37°C
 * 27) Gel purification
 * 28) * 1.2% gels
 * 29) * each well only holds about 15μL sample with large comb, so needed two gels to purify all 3 double-digests
 * 30) ** First gel leaked a little while setting, but I don't think this significantly affected the well volume
 * 31) ** Taped 3 lanes together for second gel, and this held all 60μL (50μL rxn + 10μL loading buffer)
 * 32) First gel
 * 33) 15μL phosphatased NheI/SapI pTXB1His#1 (3.2 above)
 * 34) 15μL phosphatased NheI/SapI pTXB1His#1 (3.2)
 * 35) 15μL phosphatased NheI/SapI pTXB1His#1 (3.2)
 * 36) 15μL phosphatased NheI/SapI pTXB1His#1 (3.2)
 * 37) 15μL NheI/SapI BSA PCR (from step #1 &rarr; 1.1)
 * 38) 15μL NheI/SapI BSA PCR (1.1)
 * 39) 15μL NheI/SapI BSA PCR (1.1)
 * 40) 15μL NheI/SapI BSA PCR (1.1)
 * 41) Second gel
 * 42) 10μL  1KB ladder
 * 43) 60μL phosphatased NheI/SapI pTXB1His#1 (3.3 above)
 * 44) * run in tandem; 50' @ 90V
 * 45) * stain in EtBr
 * 46) * cut out samples:
 * 47) dd Insert (1.1):
 * 48) * 1.6759g-1.0282g = 647.7mg
 * 49) * add 650μL Membrane Binding Solution
 * 50) dd Vector(His) (3.2):
 * 51) * 1.5500g-1.0275g = 523.5mg
 * 52) * add 530μL Membrane Binding Solution
 * 53) dd Vector (3.3):
 * 54) * 1.5304g-1.0320g = 498.4mg
 * 55) * add 500μL Membrane Binding Solution
 * 56) * follow | Wizard SV Gel and PCR Clean-up kit vacuum protocol instructions
 * 57) ** each sample took 2 passes through column (<700 μL each time)
 * 58) ** I failed to incubated the dissolved gel solution on column before applying vacuum
 * 59) * store @ -20°C
 * 1) * follow | Wizard SV Gel and PCR Clean-up kit vacuum protocol instructions
 * 2) ** each sample took 2 passes through column (<700 μL each time)
 * 3) ** I failed to incubated the dissolved gel solution on column before applying vacuum
 * 4) * store @ -20°C

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


 * 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.

Second gel purification



 * }