is PCR credible enough for validating my clones? - (Jan/25/2006 )
i have done a ligation of 4 fragments,electroportate it into Ecoli, and get several clones on selective plate.then verify them with PCR primers which was used to amplify the fragments above.PCR demonstates some clones are what i want.is that enough? should i extract plasmid and digest it to verify them?Thx first!
Only PCR is not enough. Always do a disgestion to verify your clones.
In our lab it happend quite often that we got wrong results in the PCRs (f.ex. all 20 clones negative; in restriction digestion 15/20 positive - and vice versa).
I think it's good to verify with RE if the construct is very important to you, but I've never had issues with using PCR as the sole validation method with the proper controls.
The issue with restriction digests is if you have a low copy number plasmid, sometimes you have to go through the extra day of chloramphenicol amplification to get a high enough yield for visualization. As always, time is money...
I agree with the others -- I'll use PCR as a screening method, but confirm by other means.
thank you all,the clone is for gene deletion in bacteria. i have got something from your suggestions.
In our lab, if it's really important,we always do colony PCR first to screen, then the PCR of plasmid, and digestion of the plasmid. But the most belivable way is sending them to sequence.