no gene after double digest of the cloned plasmid

[shivu]

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.

[biaani]

Normally I would screen more than one single colony to find one that is perfect. Sometimes weird things happen in the ligation, so it's good to have many colonies to choose between. You could google "colony PCR" for a convinient protocol of colony screening.
Anyway, since the PCR on the plasmid works, your gene is definitely there in one way or another. Possible explanations I could think of could be that one ligation site was destroyed in the ligation, so that you only see the linearized plasmid. Or that the concentration of your plasmid prep is so low so that you don't see the band - a shorter band contains less DNA and is harder to see on a gel. (It's useless to tell us that you used 15 µl of DNA if we don't know the concentration.)

I would try either (or both):
1. Cut with another pair of enzymes, pick ones that either cut out the entire insert or that cut once in the plasmid backbone and once inside the insert, to give a characteristic band
2. Sequence the insert using primers binding to the plasmid backbone, to see how the ligation sites have turned out.

[shivu]

Thanks Biaani,,

I did the whole tranformation again after repeating digestion and ligation, now i have got the cloned gene.

Thanks a lot for you help. It really helps a lot.
Regards