Saliva DNA extraction help
Posted 11 November 2010 - 01:47 PM
(note, our storage solution has Protinase K in it)
2ml of saliva solution mix
80uL of 4M ammonium acetate (we have tried using 2M as well)
incubate on ice for 10min
centrifuge for 15min at max speed
add 2mL of 95% ethanol to supernatant
incubate at room temp for 10min
centrifuge for 10min at max speed
add 1mL70% ethanol to wash pellet
remove ethanol completely
rehydrate with 500uL of TE
We can get DNA from the procedure, and that DNA can be used in downstream applications such as PCR. Our issue comes with the purity and quality of the DNA. Using nanodrop to asses our 260/280 and 260/230 values, 260/280 values usually hover around 1.7 and 260/230 are usually under 1. Any ideas for how to improve the quality of our extract?
Posted 12 November 2010 - 07:16 AM
If you can't formulate an alternate storage solution mix, you could try using more ammonium acetate and/or substituting potasium acetate, up to 1.25M or 1.5M final solution.
Substituting the 2ml of 95% EtOH with 0.7 volumes isopropanol (~1.5ml) may help.
Posted 15 November 2010 - 06:33 AM
and Protinase K
Posted 15 November 2010 - 08:03 AM