How to quantitate the pcr product from gel picturess - (Apr/09/2007 )
hey i am working on a project and i have saved gel pictures of my pcr product and now i want to quantitate/compare the expression of various sample. can any body help me for this or suggest me any tool.
You can't quantify expression from a PCR. You have to use Real Time PCR.
for rough estimation of DNA amount we use a standard picture of gel using some standard amounts of DNA like from 5 ng to 1 ug DNA
then we compare the band intensity and estimate the DNA amount roughly
i am not sure whether u will get benefit from this kind of standard picture
you could use image documentation system which could tell you the signal level for comparison, for more acurate comparision, you need a standard curve data for caculation the expression level.
This kind of quantitation is the so-called end point quantitation or semi-quantitation.
I think zouden is right. Standard PCR reactions go to a plateau, which cannot give you relative levels of starting material. I'd even go so far as to say that the best you can say for different intensities of the same product from different samples says more about inhibition of the individual reactions than expression levels of the sequence in question. Real time or quantitative PCR it must be, unless you can look at the mRNA directly by a Northern, and even then, you'll really only see how much RNA is there.