Trouble with PCR optimization_URGENT!!!

[felihaha]

Hi,

Currently I'm trying to optimize the PCR conditions for amplification of a particular gene. This is my first time doing this so I'm not sure the amount & concentration of each component to use. So far, I didn't get any results so I hope someone able to give me an idea on what to start with. Followed by, if I do not get any results what should I vary for the components? Shall I vary 1factor first or several at a time? Does it make a difference? I have only less than a month to accomlish this task soo... someone please help!

[admin]

Hello Felihaha,

I would start at the standard PCR reaction mix that is displayed by your PCR kits manufacturers instructions.

Go from there, and then you can optimize the annealing temperature of the primers.

So the most important to least:
1) Try a standard PCR with standard annealing conditions (can estimate your primer annealing conditions using various primer programs)
(usually you get a nice band right away with the standard conditions)

if not try this:
2) Optimize annealing temperatures. This is usually the most important optimization. You can do this with a gradient PCR machine.

3) Optimize difficult PCRs with PCR adjuvants such as DMSO.
DMSO can work for difficult PCRs and GC rich PCR. You can try 1%, 2%, and 4% DMSO after you optimize for annealing temperature.

Cheers!

[felihaha]

Thanks! Anyway, I'm not using any PCR kit so it's rather difficult for me to do. I'll try the methods you've given and hope I'll get the bands asap.