Problems with PCR in general - (Jun/01/2011 )
I am trying to do an overlap sequence for my ICD and a ubiquitin tag. I have ran my primers on a simulator and they work perfectly. My problems are: 1. I don't believe I am adding the right amount of template DNA to the PCR reaction. I know I need 50ng of DNA but I am getting myself confused over it. Can someone please give me a quick calculation to use to figure out the amount of uL I would need to add. So far I have been guessing with the template, probably my main problem. 2. My bands are really faint on the agarose gel, again probably from lack of template. 3. My ICD seems to not be annealing to the primers, again I believe from the template calculation. 4. Another problem I am facing is what is the rule of thumb for how many cycles to use? Thanks for the help in advance, my first time doing PCR.
ICD is internal control? I'm not quite sure what exactly are you doing and why, so it's hard to say if 50ng of DNA is good or not. Usually 100ng is standard amount put in the PCR reaction, but that may differ regarding to the application.
I'm not getting the concentration question? You need to dilute your DNA and don't know how? What is DNA concentration of your sample?
If your band is faint, you need to optimise the reaction, not only template amount, but annealing temperature, primer concentration, Mg2+ concentration, number of cycles (starting with 30). You didn't write any details about your PCR reaction, so it's hard to tell.