Urgent: should you dry PCR product - (Mar/31/2011 )
perneseblue on Sun Apr 3 05:38:48 2011 said:
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.
Could you explain this pls because I do not get it.
As I see it: the higher the PEG concentration the smaller the DNA you will precipitate.. thus keep in your sample.
So why would she lose the small DNA pieces if she uses enough PEG to precipitate?
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
Why would you lose the small PCR fragments? Is this because you add a "low" amount of PEG and thus will not precipitate the smaller pieves of DNA?
The size of the DNA fragments that stay in solution is influenced by the PEG concentration. I never thought about the chemical details behind the process, as it works very good and stable. Probably you can optimise it to keep smaller fragments in solution, but I never took the challenge to optimise the protocol. I also think that the amount of PEG used is close to the solubility limit - it takes long to get all the PEG dissolved.
It works fine - attached a fig with typical PCR products before and after PEG purification; the two short products are not included in the right gel.
That's a great picture. You even got rid of some primer dimers.
Thanks for the paper Dr H. 1975 
, don't we re-discover science constantly?
Talking about sizing, can someone explain to me how the size selection works with the SPRI beads? This stuff really puzzles me.
My link
(or maybe this subject has been discussed before?)
Maddie on Tue Apr 5 18:26:06 2011 said:
That's a great picture. You even got rid of some primer dimers.
Thats why I like this method.....I have some primers that produce a dimer signal stronger than the PCR product itself and which resisted all attempts to optimise PCR....sequences get great
