PCR problem

[ssidhu]

Hi all, I am new to this forum. I am having problem with my PCR. I am using two sets of primers to amplify PGK promoter-termination cassette from a plasmid as I need two cassettes with different restriction sites. I am using 200nM concentration of primers and 50 ng template in the PCR reaction. The forward and reverse primers in both the sets have same sequence except for different restriction site at the 5' end of primers. My problem is one set of primers is giving a perfect band but the other set is not amplifying the actual band but giving nonspecific bands at different positions. The only difference in these primer sets is the presence of different RE sites at 5' end, other than that the sequence is same. Please help me to figure out the problem. Thanks

[Flatliner]

Firstly, you need to check for self-annealing and primer-dimerization, there are several websites for this. some RE-sites such as NotI have high GC-counts, and may self-anneal with quite high Tm (my record so far is 48 deg C), Otherwise, play around with concentrations and temperatures. You can definitely go higher in primer concentration. I usually start at 600nM.

[Multiplexion]

You can increase the annealing temperature in order to reduce the number of unspecific bands.