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.