Primer Design Help: GFP primers for Arabidopsis (View forum version)


Posted 13 June 2012 - 08:07 AM

Hi, I am new here, and I would like some help/advice in designing primers for GFP in Arabidopsis GFP marker lines. I want to design a "universal" primer that would recognize the GFP sequence so that I can PCR genotype my plants that have been crossed with the marker. I know that I can technically use a selectable marker (kanamycin or basta) to identify the plants that are positive for GFP - but I would rather PCR genotype for the marker and my fellow lab mates have expressed interest in using the primers I should be designing for their own work. I've looked at several papers (that deal with PCR genotyping for GFP marker lines in Arabidopsis) that have provided the GFP primer sequence that was used - but there seems to be no consensus sequence between the papers and I am now confused as to how to go about designing primers for just GFP. Any feedback would be appreciated.


Posted 13 June 2012 - 01:32 PM

Basically, as far as I know, Arabidopsis has no endogenous GFP, so all you have to do is find the sequence for GFP and look at the primers others have used/design your own - if you can find the primer sequence in the GFP, it will probably be specific. To check the specificity you should also be able to BLAST against the arabidopsis genome - if no or low specificity hits turn up, you will be fine.