IUBio

Resuspension of P1 DNA pellet

Mike Choy mchoy at itsa.ucsf.edu
Fri Mar 19 13:08:57 EST 1999


Alex,

I've spent a good deal of time doing P1 preps and here's a few tips for you.

1) Your DNA is fine. P1 DNA is very large and I wouldn't expect it to enter
the gel more than a mm or two uncut, even on a 0.7% gel. The fact that your
digest produced some expected bands tells me that it is probably okay. Have
you tried reducing the amount of DNA in your digests or increasing the number
of units of enzyme? Don't be too disappointed if you don't get complete
digestion.

2) In your resuspension, I wouldn't ever vortex it, even pipetting it up and
down is a little harsh. I usually got good results by doing my final
precipitation in a corex, round bottom tube with a swinging bucket rotor.
This puts the pellet right on the bottom. I would just add 500µl TE and let
it sit overnight at 4 degrees.

3) Another couple things I've learned doing the qiagen preps: to increase
yield, I used to add 0.5ml 2M glucose and 2ml 10mg/ml lysozyme (mix fresh, in
TE) per 20ml of resuspension buffer P1. Also, heating the elution buffer QF
to 50 degrees seemed to work better for me.

Hope that helps!

-m


"Alex P." wrote:

> Hi,
>
> I was wondering if anyone has any tips on resuspending P1 DNA. Following a
> Qiagen purification the P1 DNA pellet was "resuspended" in water by
> flicking of the tube, pipetting up and down and leaving at 4C overnight.
> When the "resuspended" DNA was run on a 0.7% agarose gel very little
> traveled into the gel while most of it remained in the well. When
> restriction digests of the P1 DNA where performed the same problem
> occured. The enzyme cut some of the DNA and bands where visible but most
> of the DNA remained uncut in the well. Subsequently I have tried heating
> the DNA up to 50C for 30 min and vortexing but no improvement has been
> observed. Any ideas?
> Cheers
> Alex




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