Intensive primer dimers - (Feb/27/2006 )
I designed two primers for RT-PCR with Finnzymes Phusion DNA Polymerase. I was really careful to create them so they wouldn`t form dimers (with the great program PRIMER3), but the gene`s sequence didn`t leave me much of a choice. How could I change the conditions in the PCR to get rid of the dimers? I tried a couple of different annealing temps (80 °C, 77, 76, 72, 56) , but the dimers are always there. I don`t know how I should change the MgCl2 concentration f.ex. because MgCl2 is already included in the 5x HF Buffer.
Thanx for helping.
I have a couple questions, hopefully not too naive to your situation...
what is your purpose? to pop the gene out for subcloning and subsequent downstream apps? I ask out of curiousity; Phusion is all high-fidelity and stuff
more to the point, is it possible to PCR up a larger fragment (more choice in primer design) and cut it to size with RE's later? could you use genomic as a template to accomplish this, or does it have to be RNA (are you wanting a clone for expression)?
have you tried to design primers with another software, or possibly manually? have you tried to use 'less optimal' primers that wouldn't give you dimers? for example, I would maybe drop a little stringency with Tm or GC content...or alter the length...something like that...to get a pair that wouldn't form dimers? or have you already tried all this?
sounds like a real puzzle; I hope you can work it out
I need to amplify the whole gene from start to stop codon to clone it later on. First, I take 3-4 bases, then the restriction site for the RE, then the start or stop codon of my gene, then about 15 bases of the gene`s sequence. I think the self complementary lies within the gene sequence which - of course - I cannot change. Changing the restriction site`s sequence doesn`t help unfortunately. I thought maybe I could add something to the Master Mix that causes the primers to have a higher affinity to the gene than to themselves.