Strange analytical gel for miniprep - (Dec/05/2005 )
I have cloned a small DNA fragment in pCR2.1 using TOPO Kit and then purified the plasmid DNA by alkali lysis and phenol extraction and suspended the pellet in 50 ul water.
Here comes the funny part: I loaded 10 ul (one 5'th) of the plasmid on a 1% agarose gel, stained with 1ul EtBr/100ml gel and ran it at 90 volts. All my samples (very intense bands) migrated faster than the 500bp band of the DNA ladder, although the plasmid itself is 4kb. Now, I suspect it to be supercoiled, and I know it migrates faster than linearized plasmids, but THAT fast?
What could be wrong? Does anybody know if proteins, or sheer amount of loaded DNA can influence the speed? Or maybe something else?
No idea, anybody?
I was thinking that maybe the plasmid DNA was denatured, but all of it? That would require pretty harsh conditions, or not?
I appreciate your help.
I think it's supercoiled plasmid -- supercoiled plasmid is a pretty tightly packed little ball, and it'd take quite a small piece of linear DNA to match its ability to pass quickly through the matrix of a gel, especially a 1% gel.
Cut your plasmid with Ecor RI to pop out your insert (unless you added 5' restriction sites to your primers).
Thanks for the answer. I cloned the insert in the vector in order to sequence it, (160bp is too little for our on-site sequencing core to handle) so I don't want to cut it loose from the vector... I'll try with what I've got.
Which competent cells you used? use recombinase minus cells. Your clone might have got recombinated within the bacteria which gives weired looking dna on gel.!