troubles in PCR amplifying long target - (Aug/23/2006 )

hello! i'm trying to PCR amplify 3000bp from viral-integrated genomic DNA, but so far have absolutely no luck if anyone can provide any insight, it'll be very helpful!!

this is what i've done:

I've designed several forward and reverse primers and run primer combinations on viral-integrated PLASMID DNA, all primer combinations worked effectively. The next step I did is moved onto genomic DNA, but I got no band at all for a single round PCR. When I do nested PCR, I either got no band or band at completely wrong size. I've checked my genomic template by amplifying small product (300bp) which was successful. I've also increased the Tm to increase specificity, but no luck. The polymerase i'm using is Easy-A (from Stratagene), I don't know if switching to other enzyme will help at all.

Any suggestion will be extremely helpful and greatly appreciated! Thank you!!

You probably need an XL type enzyme for the 3000bp length regardless of template. Try extending you extension time to 3.5 or 4.0 min. first with your current enzyme.

If you don't see your band try to find an enzyme specifically for long pcr. Stratagene offers a couple as well.

I'm already using an extension time of 6mins.

Easy-A works well on plasmid template but not on genomic, I don't know whether it's the polymerase or the template (or both) that cause the difference.

I'm going to try Herculase II Fusion polymerase (also from stratagene), wonder if anyone has used it before and if it works well?!

I'm already using an extension time of 6mins.

Easy-A works well on plasmid template but not on genomic, I don't know whether it's the polymerase or the template (or both) that cause the difference.

I'm going to try Herculase II Fusion polymerase (also from stratagene), wonder if anyone has used it before and if it works well?!

Hi,

I am trying to amplify CYP2D6 from human genomic DNA. Has pseudogenes, so want to amplify whole genne as such. Primers are from Lundqvist et al. (1999) and generally used it seems. I`m using Roche's Expand High Fidelity PCR system. However, I'm struggling to get the right sized band (about 5kb). Tried different MgCl2 concentrations. Nothing. Protocols vary among papers and also tried various suggested PCR programs.

So what do I want to know? Anybody got any experience with this nasty gene? Anyone who can give me some suggestions on what could make a difference? For example how critical is the extenxion time (had programs suggesting annealing and extension step @68C for 6.5 min, some have different annealing temp. and extension at 10 min)? Also could adding something like DMSO make a difference?

Help a postgraduate student in need!!!!