Doing PCR on Nebulized DNA - (Apr/07/2011 )

Hey everyone,

So I'm trying out a new protocol in our lab, RAGE. RAGE is basically RACE but for genomic DNA. Step one of RAGE is to fragment your gDNA into fragments of a workable size (1-5kb). I used a nebulizer and got a really nice smear of product ranging from about 1.6kb to about 4kb. The DNA was cleaned by Phenol/Chloroform/Isoamyl and then ethanol precipitated. All the nanodrop readings came back alright so I decided to test out the quality by running standard PCR.

I used four sets of primers for unrelated gene fragments of different sizes (100bp, 600bp, 1kb, 2.4kb) to see what I could amplify. The 100bp fragment amplified, but I'm not seeing amplification of any of the larger parts, even though my smear would indicate that with random shearing I should have at least SOME of those genes contained within a fragment.

My PCR reaction was 25ul, with 50ng total nebulized DNA. I was wondering about the possibility that because my DNA is fragmented (intentionally) that perhaps when I sample 1.5ul of DNA out of my tube for the PCR, I could simply be missing the fragments I need by drift basically. It's possible I should run a higher volume reaction with more DNA?

Has anyone done PCR on fragmented DNA that has any tips about how to ensure you get a representative sample of fragments in your reaction?

Thanks all.

You should see if your PCR reaction works with unsheared DNA. The primers may not be working. I think you have too much DNA in the PCR reaction, not too little, by large factors.

phage434 on Fri Apr 8 00:21:46 2011 said:

The primers work just fine in the unsheared DNA. I ran them in parallel and I have four clean bands with unsheared, and only the 1 band (100bp) in the sheared sample. Sorry, forgot to mention that. Notably, the 600bp and 1010bp fragment amplify VERY well in the unsheared DNA (very dark bands).

Do you mean I have too much DNA in the PCR reaction because it's fragmented? I always use 50ng DNA in my PCRs...

Also, thank you for replying!