Best way to desalt very salty, low yield DNA
Posted 29 September 2009 - 06:12 PM
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
Posted 30 September 2009 - 04:02 AM
Posted 30 September 2009 - 10:49 AM
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.
Posted 11 October 2009 - 09:19 PM
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.