I have amplified my gene of interest (GOI) present in a plasmid (pET system) using PCR and sub-cloned it in pJET. Then, using restriction enzyme digestion transferred the GOI from pJET into the target plasmid (pQE80L). The E.coli clone was confirmed using colony PCR and also by doing PCR after extraction of plasmid from the clone.
(note : the plasmid after extraction, when run on a gel gave 4-5 bands. However, before cloning the same plasmid gave only 3 bands)
Moreover, double digestion of the target plasmid from the clone did not release the insert and the plasmid remained uncut (note: many combinations tried for digestion). Protein expression studies of the clone (in an expression host) too gave a negative result.
I am confused because positive result in PCR indicates insertion of the GOI into the plasmid. But, why no protein is expressed? How to troubleshoot this pbm?
Dear Aarthy, from my cloning experience I realised it's the confirmation of the insert into a plasmid it can't be trusted. I had so many types of false positive and false negative results that I end up believing that validation via PCR it's a waste of time. The true confirmations are either the release of the insert via double digestion or sequencing. If you can't release it, that the chances are that you actually have the insert in the desired plasmid are very low. You can design some primers inside your GOI and try to sequence from inside of GOI towards the plasmid. I wonder you why have 3 bands when you PCR it before cloning? Are you sure you cloned the right product? Have you sequence it? My next question is: why you sub-clone it first into pJET? Why you can't go straight to the target plasmid pQE80L? What is your cloning strategy? I could try to help you with this.
I accept the suggestion provided by Elena Daniela Aflorei. If you can able to get the answer for those above question!!!!, you will be success in the cloning experiments.
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