Urgent: should you dry PCR product - (Mar/31/2011 )

Hi everyone,

A colleague of mine is about to dry 80-100ul of PCR product in a speed-vac to concentrate her DNA. She needs her DNA in 16ul.
I'm not sure why but it seems that people do not do that. Instead they use precipitation, concentrators like centricons or ultra4, or a kit like the Minelute, right?
Do you know why people do not dry their PCR product? I've heard about severe loss? Is that true?
The colleague is about to start, so I would really appreciate your input before it's too late.
PS: the PCR product will be used for next generation sequencing.

Thanks.

Maddie

PS: the PCR product has already been cleaned-up with minelute so there is no primer and the salt concentration should be low.

I used to do that, and I had no problem with my product;The only thing is that you should be careful with how long you let it dry!.
But I did not use it for sequencing

laurequillo on Thu Mar 31 14:52:07 2011 said:

Maddie on Thu Mar 31 15:39:44 2011 said:

laurequillo on Thu Mar 31 14:52:07 2011 said:

I used to do that, and I had no problem with my product;The only thing is that you should be careful with how long you let it dry!.
But I did not use it for sequencing

Good cause..she started

The real issue is what else is in the tube when you are done. If you speedvac the pcr reaction directly, then all of the PCR buffer, enzymes, excess dNTPs, dNMPs etc. are left in the tube, but are all now at high concentration. You may not want this (usually you don't).

Hi Maddie,

I agree with phage434.

However, if I need to concentrate my PCR products prior to sequencing I use the PEG protocol below. Also use it for all standard PCR cleanups for sequencing: in my hands its the most robust and the cheapest way or PCR cleanup and the time needed is OK (also suitable for 96 well plates and PCR tube stripes, so some people from my lab now use this protocol instead of exosap). But depending on which NGS method you are using it might be not suitable: all PCR fragments smaller than 200 bp will be removed with the primer dimers - which would be fatal for Illumina; still it should work for 454 - but correct me if I am wrong.

PEG cleanup

edit: bad spelling

gebirgsziege on Fri Apr 1 10:23:16 2011 said:

Hi Maddie,

I agree with phage434.

However, if I need to concentrate my PCR products prior to sequencing I use the PEG protocol below. Also use it for all standard PCR cleanups for sequencing: in my hands its the most robust and the cheapest way or PCR cleanup and the time needed is OK (also suitable for 96 well plates and PCR tube stripes, so some people from my lab now use this protocol instead of exosap). But depending on which NGS method you are using it might be not suitable: all PCR fragments smaller than 200 bp will be removed with the primer dimers - which would be fatal for Illumina; still it should work for 454 - but correct me if I am wrong.

PEG cleanup

edit: bad spelling

It is due to the PEG precipitation step. The higher the final concentration of PEG (and higher concentration of NaCl) used, the smaller the DNA fragments that will precipitate.

perneseblue on Sun Apr 3 05:38:48 2011 said:

Here's a paper about it, Nucleic Acids Research Vol. 2(3): 383-390, 1975(!) with a draft for downloading


Link