pcr template - (May/09/2008 )
I am doing subcloning. My cDNA has already exists in one vector, I am designed one pair of primers which carries tow digestion sites and try to pcr the cDNA out using the plasmid as template. I tried several times but failed to get product. I am just wondering whether it will be easier to get product if I cut the cDNA out from the original plasmid and purify it from gel and use this fragment as template to do my PCR. I am not sure whether it will be easier for primers annealing to the fragment than to the plasmid. If it is better, please give me some suggestion about the range the amount that i should use for this kind of template.
Have you double, triple checked your primer design and PCR program (temperatures)? Is your polymerase appropriate for your product length? Do you get primer dimers, or just blank gels?
I don't think there is a distinct advantage of PCRing off a fragment vs. plasmid.
There's an advantage if you're getting non-specific fragments (by removing other possible templates in the vector) but generally that sort of problem is not the template, it should amplify well. Having said that, too much template, particularly using a minipreped clone, can result in poor amplification - the primers almost don't know where to bind there's so much DNA. If the template is GC-rich some betaine may help. And make sure the primers are correct, the annealing temperature sufficiently low and the extension time long enough.
How much template are you using? If you are using DNA directly from the miniprep first thing I'll try is diluting that, 1:20, 1:50 even 1:100 should work. If your primer design is correct PCR should work fine from plasmid, no need to digest and purify.
You can digest the vector with a restriction enzyme near the insert to make it more available to the Taq (don't need to gel purify). Make sure that enzyme don't cut your insert.