DNA or cDNA? - (Oct/12/2012 )
I need to perform an endpoint PCR to test whether i have knocked out a specific gene in the heart. My gene of interest has been floxed and subjects with this floxed gene have been crossed with cardiac specific MHC alpha cre mice and offspring genotyped.
I wondered whether i could use DNA extracted from cardiac tissue (qiagen gentra pure gene) with PCR primers for my gene of interest and MHC (in a similar manner to processing tail snips) or whether i would need to perform an RNA extraction then a reverse transcription to create cDNA then PCR. I usually do alot of realtime PCR so my knowledge of endpoint PCR in comparison is lower and wanted an outside opinion.
I also have two sets of primers for my gene of interest- <1> genotyping ones which consist of WT and flox (and also a KO primer which i do not use) and <2> primers for my gene of interest that i designed for RT-PCR. I assume if i have knocked my gene of interest out that both sets of primers would give me the same results.
I also thought that if i performed a real time PCR that i could infact obtain realtime data and then run the products on a gel also
any help, advice etc that anyone has will be appreciated
Use all 3 genotyping primers including the KO one - that way you will (probably) get 2 bands for the presence of the gene, and one for the KO. These bands are likely to be different sizes too. This should work on genomic DNA.