PCR TROUPLESHOOTING - (Oct/27/2007 )
we extracted bacterial DNA from human serum sample, we did a PCR using 16S rDAN universal primers to amplify bacterial target in the serum, we measure the PCR products after the amplification by the Spec & it was 300 ng/ul.
The problem is that we cannot see it on the agarsose gel but we see the ladder. How can I explain 300 ng/ul of PCR product but nothing appears on the gel? what do you think?
A spectrophotometer gives the total amount of DNA in the sample (primers and DNA template are included). As you see nothing on your gel, I believe all the DNA present in the sample is probably a mix of your initial primers, and non specific DNA products and template, which on a gel will be a faint smear. The gel unfortunately says that the PCR reaction has failed (for one reason or another)
I agree with perneseblue, the spec won't discriminate between product and primers so the concentration values you get are a crude estimate.
If your amplicon is small ~100 bp, then it will be difficult to see on a gel if you haven't loaded enough. If it is small, then you might need to run a high % gel i.e. 2-3%
Your PCR might have just failed? Are you able to run a positive control with your PCR or just a sample that has worked previously?
What was the spec reading for the DNA prep before you did the PCR?
As for gels, try a polyacrylamide gel and silver staining.