Double digest after cloning doesn't give gene - (Jan/07/2012 )

Dear All,

I have recently amplified one of the gene and then RE digested with NDEI and XHO. Similarly RE digested the isolated plasmid pet28c. I used NEB enzymes and used 15 microlitre of purified PCR product/isolated plasmid
0.3 microlitre of 100X BSA. 3 microlitre of NEB Buffer 4. Enzymes- 1 microlitre each and water to 30 microlitre, kept at 37 degrees for 3 hours. Then ligated them. Also made a control where PCR product or gene was not added. Transformed in DH5 alpha and spreaded on agar plate. I didnt find any colony in control, but lots of colonies in test plate. I used one of them and inoculated and isolated plasmid. Run PCR. It gave amplification of the required gene. But when double digesting it again to confirm using the same amount as i wrote earlier.
When i run gel, only I m able to see the plasmid, but no gene.
So, what does that mean, if insert was not there in the plasmid ? but having no colonies in control suggest that the insert should be there,,,

Please suggest some possible reasons and help.

Thanks a lot.

Unless you inactivate the enzymes prior to ligation, the ligated sites will be re-cut. Heat kill the enzymes if possible, or purify if necessary. This likely is one of several potential problems, but I can't tell without more details.

Dear Phage 434,

thanks for your reply..

I did cloning and to confirm if insert is there in the plasmid or not, i was doing RE digest, though i didnt do inactivation of enzymes, but my next step was not to ligate them. I just have to run the gel in which i was expecting two bands, one for the plasmid another for the gene. Do u think it is neccessary to inactivate the enzyme and the problem was due to that,,,

please help

OK, I'm thoroughly confused as to what you are doing and want to do.

hmm,, I m sorry for that confusion,,Actually I have to clone one gene. First i amplified that gene,, which is fine. Purified the PCR product. Then isolated plasmid, PET28c. Digested it with the two restriction enzymes same as that one in the amplified gene, Also digested the gene which I got after amplification. Ligated them and transformed into DH5alpha. Isolated the plasmid, did PCR using it as template. I got the band for my gene. But also to confirm i again double digested the plasmid. Run on gel. So, if an insert is there, it should cut out when i did digestion and on gel i should get two bands, one for the plasmid and another for the gene. That is where the problem is. I m not getting the band for the gene. Hope i m clear this time.

OK. It's clear. Is the band on your final gel the same size as the pET28 band, or larger? PCR is an unreliable way of determining the presence of a DNA fragment, due to high sensitivity and contamination issues. Have you run a negative control for your pcr reaction? I'm guessing that you do not have an insert in your plasmid, but that the pcr is working due to contamination problems. You could try a more reliable pcr, using one primer on the pET28 plasmid, and another on your insert. If the band is the same size as the pET28 vector, then I'd say you almost certainly have no insert.

thanks a lot phage
434, i will do this control to check for the contamination and will do transformation again. Thanks a lot for your help.