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Can't precipitate DNA!

Can't precipitate DNA! - Protocols and Methods Forum

Can't precipitate DNA! - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 10-25-2003, 02:03 PM
atgcpaul@yahoo.com
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Default Can't precipitate DNA!



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. 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.

Thanks,
Frustrated Paul
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  #2  
Old 10-26-2003, 12:44 AM
David Micklem
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Default Can't precipitate DNA!

In article <bndvqk$4thh$[Only registered users see links. ]>, <[Only registered users see links. ]>
wrote:


Wot? No ethanol? Or is it added with the NaAc?

Then it's spun at 4500g in a cold plate spinner. The aluminum

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
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  #3  
Old 10-26-2003, 04:17 AM
atgcpaul@yahoo.com
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Default Can't precipitate DNA!

David Micklem <[Only registered users see links. ]> wrote:

: Wot? No ethanol? Or is it added with the NaAc?

Ice cold 200 proof ethanol added to PCR with sodium acetate--all
separate solutions.


: 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.

Since this began, I have been extra careful with the 70%. It's 70%
made from 95% denatured ethanol--also works for sequencing rxns. In my
last attempt, I added the 70% right on top of the other mix without any
dumping. Even if I didn't wash away all the salt, the pellets weren't hit
directly with 70% and I should have still had some product.

I think next time I'll switch plates or try different sodium acetate
mix.
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  #4  
Old 10-30-2003, 05:21 AM
atgcpaul@yahoo.com
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Default Can't precipitate DNA!

I did another PCR today with a different type of 96-well plate (PE
Microamp plate) and precipitated with sodium acetate from another tech in
all the wells except the last column. In column 12 I used the stock I've
been using since this ordeal happened. After the precipitation ALL my
samples were present and at about the same concentration before the
precip. That's good enough for me that there's something up with those
other 96-well plates.

Paul
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