Double digest after cloning doesn't give gene
Posted 07 January 2012 - 12:44 PM
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.
Posted 08 January 2012 - 05:48 AM
Posted 08 January 2012 - 10:13 PM
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,,,
Posted 09 January 2012 - 06:55 AM
Posted 09 January 2012 - 10:15 AM
Posted 09 January 2012 - 12:23 PM
Posted 09 January 2012 - 09:10 PM
434, i will do this control to check for the contamination and will do transformation again. Thanks a lot for your help.