PCR

I'm back full time in Dr. Schmidt's lab. Last week was a short break for me since it was finals week and a few days of just looking through my previous results from the Schmidt's lab. Had a chance to go to Holland on Sunday with Nok, which I might blog about it later if I find the inspiration to. It was fun because we didn't plan it at all and I love spontaneity.

Ever since I came back to the lab on Monday, I've been doing nothing but running Polymerase Chain Reaction (PCR). I have to amplify a 4.5 kb long construct out from a 7 kb plasmid. I've tried a few times with different conditions and I haven't manage to see even a tiny bit of product yet.

1. Used regular Taq polymerase from Invitrogen.
2. Increased elongation time to 5 mins.
3. Used XL rTth DNA polymerase from Applied Biosystems.

Now running one with annealing temperature between 45oC to 64oC and I decreased the DNA template to only 10 ng. Hopefully at least one of these reactions will give me a product. Something for me to move on. This is only the first step in creating the broad host range construct and I'm already stuck. Really need to get this done before ASM since after ASM I would not have much time before I go home.

~frust~