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Saliva DNA extraction help


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#1 Firecam

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Posted 11 November 2010 - 01:47 PM

Alright, so we've been attempting to extract DNA from saliva using the following procedure:

(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
remove supernatant
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?

#2 David C H

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Posted 12 November 2010 - 07:16 AM

What is in the "storage solution mix"? I am guessing that it contains guanidine isothiocyanate (GITC). GITC tends to do a very good job at precipitating polysaccharides, which could be why you have a low 260/230 ratio. Guanidine HCl also precipitates polysaccharides, but no as well as GITC. Substituting the guanidine with other salts (NaCl, NaI, etc) may improve the ratio.

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.

#3 Firecam

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Posted 15 November 2010 - 06:33 AM

The storage solution is Slagboom buffer which contains
1M NaCl
Tris HCl
EDTA
SDS
and Protinase K

#4 David C H

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Posted 15 November 2010 - 08:03 AM

What is your DNA yield? If the yield is below 50ng/ul, you may not be able to do much about the purity (impurities that are masked by a high yield are more apparent at low yield). It's possible that you are bringing down a lot of salt and possibly SDS in your pellet, and that is somehow effecting your reading. I've had instances where the 230 reading after using a column-based cleanup kit was worse than it was before cleanup, and I don't know what would cause that. In addition to the suggestions above, you could try a buffer with less SDS and proteinase K, but if your downstream applications are fine, I don't know that I'd spend too much time trying to fix what may not be broken.




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