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Primer clean-up - ExoSAP-It isn't working (Mar/17/2010 )

Hello all,

We are attempting to create a SNP assay for 5 hotspots in the mitochondrial genome using an extension primer technique. We are having an issue of not being able to fully remove the main amplicon primers from the first amp product using ExoSAP-IT before the addition of the extension primers, and would like to see if anybody has any feedback. We have run the ExoSAP-IT under the recommended conditions, 37 degrees © for 15 minutes followed by 80 degrees © for 5 minutes. The product soaks at 4 degrees © for short-term storage purposes. We have also tried extending the incubation times to 37 degrees for 90 minutes, 80 degrees for 20 minutes, and we added an incubation at 25 degrees followed by the 4 degree soak. Any other suggestions?

Thanks so much!

-intern-

Use QIAquick PCR Purification Kit - http://www1.qiagen.com/Products/DnaCleanup...icationKit.aspx

-vladooo-

Or MinElute PCR purification kit which works with very small elution volumes (also from Qiagen, cat #28004 or 28006)

-Maddie-

intern on Mar 18 2010, 02:15 AM said:

Hello all,

We are attempting to create a SNP assay for 5 hotspots in the mitochondrial genome using an extension primer technique. We are having an issue of not being able to fully remove the main amplicon primers from the first amp product using ExoSAP-IT before the addition of the extension primers, and would like to see if anybody has any feedback. We have run the ExoSAP-IT under the recommended conditions, 37 degrees for 15 minutes followed by 80 degrees for 5 minutes. The product soaks at 4 degrees for short-term storage purposes. We have also tried extending the incubation times to 37 degrees for 90 minutes, 80 degrees for 20 minutes, and we added an incubation at 25 degrees followed by the 4 degree soak. Any other suggestions?

Thanks so much!

-DavidS-

Whilst there many alternative techniques I am curious about why your PCR may not be cleaned up with exosap. We have similar problems and we are wondering if this is the result of the pH of the PCR buffer...ie the pH is too high for the Exosap to work effectively. What i the pH of your buffer...also - how big is the amplicon?????

-DavidS-

intern on Mar 17 2010, 11:15 AM said:

Hello all,

We are attempting to create a SNP assay for 5 hotspots in the mitochondrial genome using an extension primer technique. We are having an issue of not being able to fully remove the main amplicon primers from the first amp product using ExoSAP-IT before the addition of the extension primers, and would like to see if anybody has any feedback. We have run the ExoSAP-IT under the recommended conditions, 37 degrees for 15 minutes followed by 80 degrees for 5 minutes. The product soaks at 4 degrees for short-term storage purposes. We have also tried extending the incubation times to 37 degrees for 90 minutes, 80 degrees for 20 minutes, and we added an incubation at 25 degrees followed by the 4 degree soak. Any other suggestions?

Thanks so much!


let us keep in touch, send me a mail hsieh@neb.com and we might have something for you to test of SNP assay.

-pei-

Hello all:

I just received at the lab a package with exo sap-it and esnt to start using it for PCR product purification.

Nevertheless, unfortunately it remain between 3-4 hours in my desk, at 24-26ºC until I arrived there.

My question is if you think it degraded because of that reason.

Thanks in advance,

DAVID

-davidomartinez-