July 1, 2009
PCR
- ran a gel of yesterday's PCR (62°C, magnesium gradient)
- Gel is mostly blank except for 035
- Looks like we didn't add polymerase and primers, but we certainly did
- Possibly the change in temperature messed things up?
- Will use 035C(2) in restriction digest to try to determine nature of the band
Restriction Digest
- BstBI/BglII double digest
- Should have 1 fragment at 333bp if we have GFP - AHI construct OR kan-ADH construct
- Ran a gel of product. Weird: No apparent digest.
Gel Purification
- PCR. Used purified product as template.
- 2 MgCl gradients (0 and 2) at 58°C annealing
- for 49, will try all 4 gradients (0, 1, 2, 4) at 55°C annealing
- After running PCR, realized we mixed up primers/templates for 139 and 186; they were discarded.
- Ran a gel; only got funky bands at 850kb; no actual product
Top: Ladder, -DNA, -Pol, 035, -, 49, 213, -, 236, 346. At 55C 41(0), 49(1), 49(2), 49(4)
Bottom: Ladder, -, -, 035, 49, 186, 213, 236, 346
 - must be a mispriming error, which is weird because they are supposedly no redundancies according to the sequence
PCR
- Realized (after running out of polymerase) that we've been doing our reactions all wrong. Correct recipe is below. Will have to order more polymerase and/or use another kind.
- Correct PCR recipe using Accusure polymerase (20μL RXN)
10μL of reaction mix
10X NEB buffer.. 2μL
dNTPs..........0.5μL
DNA............0.5μL
Primer 1(5μM)... 2μL
Primer 2(5μM)... 2μL
ddH2O........... 2μL
Polymerase.......1μL
10μL of H2O/MgCl mix (concentrations of MgCl at 0, 1, 2 4). Use 20mM MgCl
MgCl......ddH2O
0: 0.......10μL
1: 1........9μL
2: 2........8μL
4: 4........6μL
*Add reaction mix to H2O/MgCl mix at whatever MgCl concentration you desire. Total will be 20μL
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