3 replies to this topic

[DaisyCarbine]

One of my labmates is trying to amplify a sequence from a plasmid that we have received from another lab.

Her PCR has been failing, and there seem to be some issues with the template itself (i.e. the plasmid). PCR produces a very faint band at 12 kbp. The plasmid is supposed to have a MW of 7 kbp. I ran a gel checking the plasmid for size -- I obtained a very intense band at 7 kbp. Other labmates have checked larger quantities of plasmid on the gel; they have seen the 7-kbp band but also seen a smaller contaminating band at roughly 12 kbp.

I ran a gel containing some of my labmate's PCR attempts, a sample of undigested plasmid, and a sample of linearized plasmid. PCR produced the same wrong 12-kbp band. Furthermore, the 7 kbp band has completely disappeared from the samples that should have contained plasmid, and the 12 kbp band has gotten much, much more intense.

Clearly, we need to go back to the glycerol stock of this plasmid and make a new prep. But I don't understand what has happened to our stock of plasmid, and I'd like to keep this from happening in the future. Any ideas?

[bob1]

Were there different preps of the plasmid between gels?  If so, you may have mostly supercoiled DNA in the prep, which could be running at the 12 kbp point.

[DaisyCarbine]

It's the same prep used in every gel. Also, I digested it with restriction enzymes -- shouldn't linearizing the plasmid get rid of supercoiling?

[mdfenko]

DaisyCarbine, on 06 June 2012 - 06:34 AM, said:

I digested it with restriction enzymes -- shouldn't linearizing the plasmid get rid of supercoiling?

yes