3 replies to this topic

[Micro]

Hi,

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?

Cheers M  

[Adrian K]

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...


good luck.

[molgen]

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.

Edited by molgen, 29 November 2009 - 07:20 AM.

[Micro]

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!

Cheers
M