Can't precipitate DNA!
drmicklem at nospam.com
Sat Oct 25 19:44:09 EST 2003
In article <bndvqk$4thh$1 at netnews.upenn.edu>, <atgcpaul at yahoo.com>
> I have a perplexing and embarrassing situation at lab--I can't
> precipitate my PCR products! We're a high throughput lab so our
> PCRs are done in 300ul PCR plates. The PCRs work fine. I do a
> standard sodium acetate/ethanol precipitation in the plate. I
> do it EXACTLY like the other 2 techs at work who have it working.
> There are 2 differences, though, which I haven't explored yet.
> Our PCR plates come from the same company but they were are shipped
> in separate boxes of 50 plates. I happened to grab this box and they
> grabbed whatever box they got. The other difference is we each have
> our own sodium acetate. However, it was made in the same beaker and
> split into 3. My stock is also used for making the sodium acetate
> mix for precip sequencing rxns which works fine (those are done in
> 384 plates).
> It's not that the precipitations completely fail, it's that most of
> them fail but not all of them and in no pattern. The sodium acetate
> solution is added to the PCR reaction and it's incubated in the -80 for 15
Wot? No ethanol? Or is it added with the NaAc?
Then it's spun at 4500g in a cold plate spinner. The aluminum
> foil tape is pulled off and the plate gently inverted over the sink and
> wiped off. Then 70% ethanol is added and the plate is spun again at 4500g
> for 15 minutes.
Could it be that you are squirting in the 70% ethanol more vigourously
than the others and dislodging the pellet? I never think they stick as
well after being dislodged, even if you spin again.
The plate is gently inverted over the sink and then
> invert spun to 50g. The samples are dried and we proceed with a digest
> overnight and then another precipitation followed by a ligation.
> 2 days ago I had samples carry through both precipitations although I
> lost a small amount. The next day I ran a gel after my first precip, and
> it was great. I did the digest, precipitated, and I lost nearly every
> sample! I repeated the PCR. precipitated, and lost almost everything
> again--after the first one. It's not that I lose everything--there are
> always a few stray wells that work fine and they aren't always the ones
> with the most product.
> If I lost everything after the digest, I'd suspect I had DNase
> contamination or something but that's not the case and I don't lose every
> sample. It's very annoying. Could it be that my box of plates has more
> slippery walls than the others? I try not to let the precip/DNA mix touch
> the foil tape adhesive when I mix but I've done it in the past and it was
> fine. Until this past week, my precipitations have been fine.
> Frustrated Paul
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