Increasing the number of products your PCR produces - Degenerative primers for multiple copy gene (Nov/23/2009 )
I have degenerative primers for a gene that occurs upto 8 times in my genome. Each copy of the gene has varying specificity to the primers resulting in the dominance of a couple of products using my current method...but I want to amplify all or at least most of the genes.
Does anyone have any suggestions for decreasing the specificity of primers?
I am considering things like:
<*>Increase annealing duration
<*>Decrease annealing temp.
<*>Put in additives
<*>Remove additives (I'm currently using DMSO)
<*>Use normal Taq not HotStart Taq
<*>OR (as a last resort) design two set of primers to target a smaller group of genes
Does anyone have expereince with these option working or not working, as the case maybe?
I used to have idea of amplify multiple sets of genes in one shot...however after many failure, I stick back to do it one by one or couple by couple....to kill 10 birds with one stone, i throw the same stone 10 times.
You can still try what you had consider, who knows it might work...
Though the best way, in my mind, to go about this is to aligning all of the transcripts and to pick primers for the conserved regions, I can't see what good it will do you.
I think that the better science is to evaluate them separately (3'UTR for instance) and then sum them up.
Thanks for the feedback.
I do agree that getting all the genes is asking a lot.... however, IF I can get primers to pick up multiple genes I'm going to be using them for T-RFLP. For this reason having 1 or 2 sets is really the only option, since the fluoresent marker cost too much to get 10 primers made up. Not to mention that I don't have the genome sequence for one of the species I'm working with, so I am throwing stones while blind folded in some cases.
But that is the fun of science, figuring these things out!
I will look into the UTR suggestion as an alternative to conserved regions, which I am currently using.
Anymore comments or suggests are more than welcome!