double digestion not working - (Feb/04/2009 )

hi
I had performed one ligation and got a single colony after transformation.im trying to isolate plasmid from the ligated colony but the yield is very low and also double digestion of the plasmid is not working.But surprisingly the plasmid is showing pcr confirmation of the presence of the gene cloned in the plasmid.positive control has a band and the ligated sample also showa band at the same region.how to solve the problem of this digestion(using ecoRI and hind III)plz can anyone help ?

sagar on Feb 4 2009, 11:19 AM said:

Is it possible that your PCR has contamination? If you can't see the plasmid on gel and insert on digest, you should forget about this single colony and repeat the ligation. IMO.

TanyHark on Feb 4 2009, 08:47 PM said:

I agree with TanyHark. The restriction digest is more definitive than the PCR. PCR is very sensitive, if you use PCR to confirm plasmid construction, always amplify across the junction between the vector and insert (one primer binds to the insert and the other the vector). If you don't amplify across junctions, PCR can amplify left over DNA on the plate.

could you confirm the enzymes work? Do a positive control digest next time just to make sure.

hello

actually pcr is not having any contamination im sure of that the problem is that initially when i isolated the plasmid and carried out pcr with it along with the positives control the band obtained were exactly at the same position.the primer is gene specific.im quite sure about the presence of the gene in my vector.i think that some base pairs might have got affected for the digestion enzyme thus its not working .
thanks anyways