Can't precipitate DNA!

David Micklem 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>
wrote:

>    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
> minutes.  

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.
> 
>                                 Thanks,
>                                 Frustrated Paul



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