PCR primer question

[duga]

Hi, guys,

I'm surprised I haven't joined a science forum until now. Leave it until I have a problem. Sorry if making this a new topic is bad form...I just felt I had a pretty specific question.

Anyway. So I'm trying to isolate a specific gene (end product should be roughly 3.5kbp) from Arabidopsis genomic DNA. I have gone through many a primer and I am yet to have any product. I am starting to freak out because I've been at this for a few weeks and my PI is starting to get impatient (as well as incredibly busy...I can never find him to ask questions like these).

My positive control works so it's not the DNA or the dNTPs. I switched from PFU Turbo (since I want high fidelity for further experiments) to a cheaper Taq master mix (just until I sort this out) where I only have to add the template, the primers, and some water so the chance that I screwed something up in the mix is unlikely. I performed a touchdown PCR to no avail. I've tried multiple annealing temperatures for each primer. Still nothing. I've extended and decreased extension time, annealing time, and melting time with still nothing. I've messed with concentrations...it feels like I've tried everything. It looks like it's back to the primer drawing board.

My question, then, is are there any tips any of you can give me for amplifying a gene from genomic DNA? Barring the usual GC content/no hairpins/gc clamp, etc. Has anyone tried something similar with good results? Thanks!

[JackBean]

How can you have positive control? How does your primers look like? Do they have some overhangs? How long, what Tm? What about some nested PCR?