Absolute quantification using Real Time PCR - (Apr/25/2010 )
Im a fresher to Real Time PCR and i have to determine the copy number of a gene. i have the following queries
1 I have done the reverse transcripton PCR and have got the cDNA. Now how do i construct standard curve? do i have to use log 2 old diluted plasmid DNA containing the gene or the cDNA itself?. and how do i calculate the copy number of the gene?
2. as with the plasmid, should i use the plasmid construct using the full lenght gene (2 kb) or should i clone the gene using the primer designed for real time PCR (PCR prodcut size 55 bp)?
1. The ideal way to prepare a standard curve is to make a serial dilution of your template, with each dilution being a 10-fold dilution. Ideally, the template you use for your standard curve should be the same as your samples, so if your samples are going to be cDNA, then your standard curve should also be cDNA. Calculating the copy number of your gene is a complicated calculation. Send me a message and I can email you an Excel file you can use for this purpose.
2. You can use the plasmid with the full length gene, but as I mentioned above it is best if you use cDNA
1. I once created an online aplication to calculate copy number.