Digestion of a plasmid - (Mar/27/2009 )

Hey everyone,

I ligated my gene of interest (TLE1) into a plasmid (pcDNA3.1+) and cultured the transformed bacteria on an agar plate getting about 100 colonies. I also had a control with just the plasmid ligating to itself. This control provided no colonies.

I then digested the plasmid with the insert in 2 seperate ways:

1. to linearize it with one enzyme (Hind III)
2. cutting out TLE1 with two enzymes (BamH I Xho I)

Both of the gels I ran seem to show only the plasmid was present however, with bands being at just 5.4 kb (ie not at 8kb or 5.4kb and 2.6kb)

So if the plasmid seems incapable of ligating to itself, why am I getting growth on the insert + plasmid plate yet no bands present where I expect?

Thanks

jmoran on Mar 27 2009, 04:34 PM said:

Hi

how much plasmid are you digesting? It may be that the concentration of the insert it too low to see it properly under UV light, try digesting more plasmid.

Alternatively, in our lab we routinely PCR screen our colonies by diluting 1 colony in 10ul of water and using 2 ul of this as template in a normal 20 ul PCR reaction (eg taq polymerase) but increase the initial denaturing time to 5 min to ensure the bacteria are lysed. To screen for the insert, use one primer that will bind to the vector (eg forward primer could be T7 promoter primer, which is present in pcDNA3.1) and one primer that will bind to your insert (eg the reverse primer you used to pcr the insert in the first place). You should get a pcr product of around 2.6kb if you do have insert!

Again, you could just sequence your plasmid and find out what gone on that way!