I'm interested to know whether cutting a DNA plasmid at two sites rather than at a single site will make much of a difference to the resultant qPCR amplification. The restriction enzyme I've been using for agarose gel analysis completely excises the target insert by cutting either side of it, and I don't fancy paying for another enzyme to do a similar job. To my mind, two cuts would mean you'd end up with the plasmid cut into two sections rather than one, but the overall DNA content would remain unchanged. I'm fairly new to absolute qPCR, so any ideas would be great.
I agree with you that cutting with one or two enzymes should not make a difference as long as the primer is still able to prime the target. Are you running into trouble or are you asking a hypothetical question?
At the moment it's a hypothetical. I really just wanted to see if I'd missed something fundamental before using several hundred $$-worth of reagents! I'll be trying it in the next few weeks...
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