All is Revealed but...

I was not satisfied with the plasmid digested with SepI-HF because it yielded different profile with that of the original one I obtained. Hence, I treated the plasmid with NotI-HF and finally the same profile was procured again. I was so gay to see the profile and immediately performed the PCR to amplify the CT region that I have been so looking forward to get. After two and half hours and ran on the 2% gel, I still got NOTHING on the gel..but WHY?? This baffled me a lot and I wondered now if the insert is still in the plasmid or not..I need to get down into this from scratch again..

                                                Where is the CT band after all the pretreatment?