Second round of PCR after gel extraction fails miserably - (May/12/2012 )

Hi all, I am trying to amplify 3-5 kb fragments from a 200 kb BAC construct containing mouse genomic fragments.
I am using primers with matched GC content and TMs (59-60C).
Running touch-down pcr that starts at 68C to 55C in 1 degree increments with a final 15 cycle amplification at 55C.
I have tried Accuprime PFX and Phusion.

I will get a light band of the correct size, with other products thrown in there, so I load the 45 uL remaining of my 50 uL product onto a gel, see the band, cut it out and gel extract using zymo's gel extraction kit.
I'll get concentrations varying from 20 ng/ul to 150 ng/ul yay!
Since I need far more for my downstream application, I take 1 uL of the gel extracted product and run a second round of PCR using the exact same conditions.
I get *only* the smaller bands which appeared in the first gel, but without any of the correct product.


What...is going on? o.O I am at my wit's end here


Summary: Run a pcr, gel extract a decently bright band. A second round of PCR on this product yields only smaller bands and not the original extracted band.

Has anyone else run into this?

Thanks!

I have never been able to use pcr products from one reaction as a template for a second round without this sort of problem. I'd strongly recommend using nested primers or at least semi-nested primers to solve the problem.

Interesting, so in your experience, semi-nested primers for round two solves this problem? o.O Dare I ask if anyone knows why?

It's things like this that make me wonder if PCR actually works the way we think it works

Thanks for the suggestion! I'll be ordering primers soon enough

I've successfully used gel extracted PCR products as template for a second round of PCR, though nothing as big as 3-5 kb, and only once or twice. It looks like you're doing about 30 cycles in your first PCR? What about just increasing to 35 or 40 cycles, maybe then you won't even need the secondary PCR?

Otherwise nested primers sound like a good idea. Without nested primers, the primers for the second round of PCR have to bind right at the very end of the template. Maybe a bit of degradation knocks a base pair or two off the end making binding much less efficient (I'm just guessing here). I know that restriction enzymes don't bind efficiently right at the end of a DNA strand, maybe primers don't like to either (more guessing ).

I read in a forum a while ago that someone would take a needle and poke it into the gel into the PCR product band, then just stick the needle tip into the PCR mix for the second reaction. Enough DNA would be transferred just on the needle tip to reamplify it. I've never tried this so I can't comment on how well it works.