restriction enzyme site with primers - (Jan/10/2008 )
i am trying to amplify promoter region from genomic DNA but could not do so. The product size is 4kb. I tried different conditions of pcr and checked all the components. Igot multiple bands of different size but not of 4 kb. I have a question that can restriction enzyme sites included in primers affect pcr.Please help me.
thanks in advance
Did you check your primers by annealing them with the promoter region?
Sometimes doing PCR off genomic DNA is just difficult in general. Are you using DNA isolated directly from the organism, or is it DNA in a bacterial clone? A clone would be much easier to work with.
try to amplify using a set of primers designed in such a way that they are just outside the ORF. Then once you get the right sized pcr product, amplify with the primers that have the restriction sites. This could help.
Can you also give seq, information about your primers?
I guess since ur primers have restriction site and also should carry extra overhang to support digestion. If so, u would naturally do not have 100% complimentarity. By manipulating the PCR conditions, it is easy to get desired product size from a plasmid, but often problematic to get from genomic DNA, due to the high sequence complexity, that can skew up ur primers binding else where. several alterations can be tried. Can u Increase the primer length for high specificity; do first two cyles at annealing temperature at lowest stringency (Tm-5C), next two cycles at higher stringency (Tm-3) and rest of the cycles some more higher. Keep your extension time for four min since ur product is bit larger (also use high fidelity polymerases for larger fragments).
check this site, http://www.med.yale.edu/genetics/ward/tavi/Trblesht.html