1 reply to this topic

[KimWG]

Hi all,

I am trying to extract and amplify insect DNA from bat feces in order to characterize bat diets.  I usually use a Qiagen DNEasy kit with no problems, but every once in a while I hit a snag -- no product from the amplification, sometimes due to extraction of inhibitors.  (Not always due to inhibitors -- but certainly sometimes).

Now, before you say "you shouldn't have inhibitors if you used that kit", well, it ain't necessarily so.  If I combine some of my problem template with my positive control template, suddenly -- poof! -- no product.  I have tried diluting my extractions (by 10X, 100X, 1000X) and this rarely helps -- the DNA is already degraded and generally low copy number, and I rarely hit that sweet spot between just enough template and avoiding inhibitors, even with extended cycling.

I know from past experience that samples that failed with the Qiagen kit will often succeed if I use an Omega E.Z.N.A. kit, which includes a chloroform extraction.  Why not use that kit all the time?  (1) I am lazy, and they don't call it dnEASY for nothing.  (2) Omega kit is backordered and right now all I have is the Qiagen kit.

I'm wondering though... is there a way to "rescue" my Qiagen extractions or remove the inhibitors?  If I performed a good old fashioned phenol-chloroform extraction ON MY EXTRACTION would this remove the inhibitors?  Or is this just crazy talk?  Is there a way to get the inhibitors out of my extractions after the fact?  Any and all suggestions will be appreciated, I am pretty new at this stuff.

[bob1]

The answer is... maybe... because it depends a bit on what the inhibitors are, having come through one extraction, they may well come through another, but are likely to be diluted/partially removed with each subsequent extraction.

I say give it a go, what do you have to lose, they can't get any more not working than they are already.