3 replies to this topic

[jessh]

Hi All,

I am looking for some suggestions about removing high concentration of salts from my DNA samples. I have been using EtOH precipitation to desalt my samples, however, my QPCR reactions are still inhibited indicating that not all the salt is being removed in this protocol. For the EtOH precip I am using NaAc and 70% EtOH for washing, as well as glycogen as a carrier.

Another consideration is very high recovery.

Columns generally work well for my routine applications but for this method I have low amounts of DNA - pg amounts. Are there any columns out there that are recommended for low amounts of DNA? Millipore may have a column that might work - the YM-100. Has anybody used this?

Or this kit:
illustra GFX™ PCR DNA and Gel Band Purification Kit

Has anybody tried the millipore drop dialysis method for cleaning up DNA?

Would there be any advantage to using organic extraction i.e. phenol/chloroform extraction?

Any other suggestions would be greatly appreciated

Thanks
Jess

[phage434]

Have you tried simply reducing the volume of DNA added to your PCR reaction? PCR will exponentially amplify your DNA, so the amount you start with is almost irrelevant, and dilution can help with many problems. If I needed to desalt, then drop dialysis on a Millipore VSWP 0025 membrane (shiny side up) floated in TE in a petri dish would be where I would start.

[ShannonJ]

Another easy way is the Microcon columns...you essentially spin out your buffer leaving the sample on a filter, then add some volume of water (or other buffer) and invert the filter into another tube. It only takes minutes but generally works well.

However I have to agree that dilution is often the solution to PCR. I've never used template that is less than 1:100 diluted in a successful qPCR reaction.

[jessh]

Thanks for your suggestions!

I tried diluting my samples for the QPCR, however, this was not enough to prevent PCR inhibition.

I have since tried the millipore drop dialysis membranes and they worked a treat! They are really easy to use, although you don't recover all of your sample.

I am waiting on the millipore columns to arrive so that I can compare those. These would be preferable for larger volumes that I want to desalt. The limit on the millipore membranes is around 200 µl.

I am also wanting to recover as much as possible - not just enough for PCR - so it will be good to see if the columns out perform the drop dialysis membranes.

Thanks again. I appreciated your comments.
Jess