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
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. 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. 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.