Can I use Real-time PCR instead of Southern Blot to determine number of integran - (Jan/23/2013 )
I have done a transfection in a hard to transfect cell (which took me several months) and now I need to screen clones for number of integrants. The manufacturer protocol recommends using Southern blot technique as it would allow me to determine which clone has only one integrant (which is what I need), however I have been trying unsuccessfully to perform a Southern for over 2 months now using a DIG labeled probe and following Roche protocol. I really don't know what's going on... have tried 2 different probes and many variations through the protocol but nothing works (never got a band, not even for the genomic DNA positive control, however I do get a band for the plasmid positive control). I know my gDNA has the integrated plasmid as qualitative PCR shows me a very clear perfect band. I was just wondering if I can use Real-time PCR (taq man) to determine the number of integrants copies in such a precision (I really need to be sure it's only one integrant not two or more)
Please, help me... the time is rushing by and I need this cell for all my project experiments!!!
Potentially you can as qPCR is supposed to be sensitive enough for this sort of thing. However, the difficulty lies in establishing controls for determining if only one copy is present - how efficient is your PCR etc...
Southern blotting is the only definitive way to determine this. I have found the Roche DIG protocols very reliable. If you are getting the plasmid positive showing up - try a dilution series to see how sensitive your blot is. It is possible that the insert isn't present, though if you have bands off a PCR this shouldn't be the case unless there is a target site for the primers in the genome (try primer-BLAST).