Visualization of pcr product before sequencing - (Sep/13/2005 )
I did a pcr to detect potential mutations in my novel gene. I started with 20 ng of genomic DNA in a 50 ul reaction. After PCR I took 2 ul of the sample and ran it on an E-gel and didn't see anything. I guess that makes sense E-gels don't have too much sensitivity--Right? These primers have always worked before, so its not the primers. Do I need to see the product on a gel before giving it for sequencing? Just wondering what folks usually do
Always check on a gel wether your PCR worked or not.
You never know if anything went wrong with your PCR, pipetting errors, bad quality of DNA, your polymerase is inhibited for whatever reason, ...
Just put some more on a gel, and if you fear you don't have enough PCR product to sequence it, just do your PCR again.
Thanks for the reply. I had forgotten to mention that I had been given a nested primer for that sequence as well. Is it possible that when I do the second round (starting with lets say 10ul of that initial 50 ul rxn), I should perhaps see a band (given that I load enough) on the gel? Its probably very hard to visualize a band starting with 20 ng of Total Genomic DNA without doing a 2nd round with a nested primer--Right? Is that one of the reasons that people use a nested-primer approach? My issue is that I don't want to waste my precious tumor DNA running it on the gel--I'll have nothing to give for sequencing!
after the gel runs, could you cut out and purify the band, then send it off? I know you lose some of your yield, but then you know you have product and you are not sequencing water
There is no reason that you should not see PCR product after a first round PCR, regardless of the amount of genomic DNA you start with. The PCR amplifies the amount of DNA, and the amount of product depends on the volume of the PCR reaction, not on the amount of template added. If you run a sufficient number of cycles and have good primers, you should be able to amplify and see a band with a single PCR. Besides, you shoulld probably gel isolate the band prior to sequencing in any case, so running the PCR results on a gel is the logical next step.
Second round (nested) PCR is important if you have problems with the selectivity of the PCR reaction -- multiple products from the first round primers, which need to be further specified as containing the second round primer(s).
Well, when I do single PCR starting from say 100 copies (in a 50 µl volume) I don't see any product, but when I do a nested PCR on this one, I get to see product. Also, when starting with less DNA as a template I clearly see the bands getting fainter (even on a nested PCR), so I believe the amount of template does mater.