unexpected band in PCR with plasmid - (Jan/10/2013 )
i have designed a set of primers for checking cloning in the plasmid pET28b. Though the primers are not expected to bring out any band with empty plasmid, it shows a band around 100bp which is also observed in the transformant plasmid. the primer sequences are:PET F- ATTCGGATCCACTAGTTATTG PET R- AATTCCCCTCTAGACCCTTGA. can anyone pls refer any online tool to get the sequence of the amplicons on submitting the template and primers.
It is most likely just primer dimer, could you post a gel photo?
Your primers both have fairly long strings of homology with the
Then if you do a primer blast (http://www.ncbi.nlm....s/primer-blast/) using just the perfect base paring parts of the primers, you get a product of 154bps.
Primer pair 1
Forward primer ATTCGGATCC
Reverse primer AATTCCCCTCTAGA
Product length 154
Actually the primers were designed earlier by my labmate, and while checking i saw the complementarity of the forward primer with the plasmid. But i couldn see the complementarity of the reverse primer, so only i proceeded with the PCR.
The secret to finding your reverse primer in your sequence: http://www.bioinformatics.org/sms/rev_comp.html