Initially we are getting positive amplification .The positive colonies are selected and restreaked.The PCR is not showing positive amplification.
Assuming Ag colonies were grown in media containing antibiotics (kanamycin), it would be prudent to double check the kanamycin stock, as well as nptII gene by PCR.
A few possibilities. One would be that you had mixed colonies (or cells with mixtures of plasmids). So they initially tested positive because there was the plasmid you sought mixed in. But upon restreaking you might have some colonies with the vector only and some with the insert. Be sure to try a number of clones to test (such as 5-10) instead of just 1 from each independent transformant colony.
Alternately it could be that you never got the proper clone at all but in the initial screen your PCR amplified some carryover DNA from the ligation that was never actually ligated to the plasmid.
I hope you were working with pure colonies. In addition to checking antibiotic, and using multiple colonies, remember to validate your PCR. For that you can include a positive control, a PCR reaction with previously validated template having your gene of interest (DNA from wild strain or wild colony itself depends on your PCR method).
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