Plasmid supercoiling affecting PCR? - (May/27/2009 )
Hi all, first time poster.
I'm using TaqMan PCR (digital PCR in particular) to amplify and detect two targets on the same plasmid. For some reason, one of these targets always seems to have twice the amplification of the other. (Digital PCR, I get twice as many hits from one target than the other for a given concentration of plasmid)
I'm wondering if the plasmid might be supercoiled or form some sort of secondary structure and hide the binding site for my probe or one of my primers? Is that feasible?
If so, what can I do to get around this/prevent it? I would think perhaps a high salt concentration might cause the DNA to unwind or something of the like, but I can't find any papers on it handy.
Cheers
Keith
kmewis on May 27 2009, 12:32 PM said:
I'm using TaqMan PCR (digital PCR in particular) to amplify and detect two targets on the same plasmid. For some reason, one of these targets always seems to have twice the amplification of the other. (Digital PCR, I get twice as many hits from one target than the other for a given concentration of plasmid)
I'm wondering if the plasmid might be supercoiled or form some sort of secondary structure and hide the binding site for my probe or one of my primers? Is that feasible?
If so, what can I do to get around this/prevent it? I would think perhaps a high salt concentration might cause the DNA to unwind or something of the like, but I can't find any papers on it handy.
Cheers
Keith
Linearized it. restriction digest
kmewis on May 27 2009, 11:32 AM said:
I'm using TaqMan PCR (digital PCR in particular) to amplify and detect two targets on the same plasmid. For some reason, one of these targets always seems to have twice the amplification of the other. (Digital PCR, I get twice as many hits from one target than the other for a given concentration of plasmid)
I'm wondering if the plasmid might be supercoiled or form some sort of secondary structure and hide the binding site for my probe or one of my primers? Is that feasible?
If so, what can I do to get around this/prevent it? I would think perhaps a high salt concentration might cause the DNA to unwind or something of the like, but I can't find any papers on it handy.
Cheers
Keith
I find this highly unlikely given that you can efficiently PCR from intact genomic DNA which is much more of a mess in terms of secondary structure.
I suspect the primers or their Tm to be the cause of this effect. PCR from plasmid is usually incredibly efficient.