13 replies to this topic

[Maddie]

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.

Edited by Maddie, 31 March 2011 - 06:51 AM.

[laurequillo]

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

[Maddie]

laurequillo, on 31 March 2011 - 06:52 AM, 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  

[laurequillo]

Maddie, on 31 March 2011 - 07:39 AM, said:

laurequillo, on 31 March 2011 - 06:52 AM, 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  

[phage434]

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).

[gebirgsziege]

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

Edited by gebirgsziege, 01 April 2011 - 02:25 AM.

[Maddie]

gebirgsziege, on 01 April 2011 - 02:23 AM, 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

The PCR product they dried is actually a mixture of several PCR products. Some had been cleaned with the Minelute before and the others with SPRI beads, so there shouldn't be any salt, dNTP etc..

Why would the 200bp fragments disappear  

[perneseblue]

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.

[Maddie]

perneseblue, on 02 April 2011 - 09:38 PM, 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.

Hmm, then it's no good. Some of the amplicons are <150bp (and mine are even <70bp). It's really hard to find a way to concentrate very small DNA fragments without losing a big portion, isn't it?
Isopropanol precipitation has been recommended to me but I fear it would cause a big loss of DNA. Same thing for cutting gels.  

[hobglobin]

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


Link

[josse]

perneseblue, on 02 April 2011 - 09:38 PM, 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 01 April 2011 - 02:23 AM, 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?

Edited by josse, 04 April 2011 - 10:46 AM.

[gebirgsziege]

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.

Attached Thumbnails

• 

Edited by gebirgsziege, 04 April 2011 - 11:47 PM.

[Maddie]

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?)

[gebirgsziege]

Maddie, on 05 April 2011 - 10:26 AM, 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