• 0.8% gel: 50mL of 1x TAE, 0.4g agarose, prestained with Sybr Safe
• out of 10 reactions only 1 reaction worked… hmmm. And there is a chance that the product isn’t even what I want. It appears that the band is not 4kb, but the gel didn’t run evenly so there is a chance it is 4kb but doesn’t appear that way because of how the current pulled the DNA.
• It looks like a machine test is in order. The Thermo cycler said the program completed today (which it hadn’t in the past few trials), but also said the program completed in 2.5 hours (which it shouldn’t because it is a 4.5 hour program). And the OpenPCR won’t get below 16C for the final hold even when I set it at 15C, which it should not have a problem holding at (I’ve seen it consistently get to 12C).
• I will also order new dNTPs even though I think that may not be the problem.
• I also learned yesterday that the freezer wasn’t closing completely and everything may have thawed potentially ruining the enzyme.
• Finally I should try to mix the master mix better if that is an issue at all.

Lots to do in so little time… Gotta get this working by this weekend.