optimization of PCR conditions - to know that primers are specific and detect copy number (May/08/2008 )
I am trying to genotype my potential mouse founders. I understand i need to optimise PCR conditions, so first
1. I want to standardize the primer conditions, if i use any genomic DNA and do a PCR with my specific primers, I am not suppose to get any band ( as the genomic DNA is not ffrom my transgenic),
a) then what do i expect in results (on gel)
and how this will help in primer-standardization ?
2..I know one can detect the copy level also by PCR, how to standardize the PCR conditions to know that it is sensitive enough to detect the transgene at a single copy level?
3...Can anybody share a picture of genomic DNA ran on a gel, its a big DNA (compared to plasmid, which i have worked on), how does it look on gel..a smear..a clear band like plasmid DNA...
Any input is appreciated.
1. if the primers are designed for your transgene, yes, you shouldn't get any band when using wild type DNA samples. when you designed your primers you should have noticed the annealing temperature, depending on the size of your product it will be the extension time (usually at 72ºC).
2. to detect copy level the besto would be by southern blot.
3. your DNA should look like a band almost at top on a 0.8% agarose gel after running a while, when your marker has run enough. a smear would imply your DNA is degraded.