I have been trying to perform absolute qPCR with a number of genes performing nitrogen transformations in prokaryotes. I've had trouble getting good efficiency (>90%), even after experimenting with different annealing temperatures, etc. I read elsewhere that it is a good idea to linearise the control plasmid first. I'm using Promega pGEM-T Easy vector.
How do I decide which restriction enzymes to use (apart from finding enzymes that cut only once, outside the target region)? Does it matter whether the restriction enzyme produces blunt or sticky ends, 5' or 3' overhang, or what temperature it cuts at? Or should I just buy a lot of enzymes and try them all?
Hi Eric,
just take an enzyme which cuts the backbone and which has ideally no star activity. If it cuts blunt or sticky is not an issue here. I would gel purify the linearized product, perform a quantification by UV VIS and calculate the copy number. Please keep in mind to keep the stock concentration high enough and to use an appropriate dilution buffer e.g. Nucleic acid dilution buffer. This is also a key point as you otherwise might loose DNA adhering tothe walls of your tubes. And finally try to optimize your protocols e.g. by Temperature gradient
Sven
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