curiouser at ccmb.ap.nic.in
Sun Jun 6 12:42:29 EST 1999
I actually removed probes ( both large as well as oligomers ) from dried
membranes. Just boil around 250 ml 0.5% SDS in a microwave and pour it on
top of the band directly. Repeat if required. In this way I successfully
stripped and reuse the blot atleast 3 times, even from dried nylon
Malay Kumar Basu
Center for Cellular and Molecular Biology
I N D I A
Oxymoron: Rush hour.
curiouser at ccmb.ap.ni.in
----- Original Message -----
From: "David L. Haviland, Ph.D." <dhavilan at IMM2.IMM.UTH.TMC.EDU>
To: <methods at hgmp.mrc.ac.uk>
Sent: Friday, June 04, 1999 7:32 PM
Subject: Re: probe stripping
> At 05:14 6/4/99 -0700, Halina Sobolewska wrote:
> >Can someone suggest ways of stripping oligo probes from dried nylon
> >membranes. I have tried boiling them in water containg 0.1% SDS. Should
> >membranes be kept wet and if so, why?
> Yes. I came across a wonderful method in the Clontech manual that, in my
> hands, works very well for both Southerns/Northerns and has worked the
> times I've tried with oligo probes.
> 1) To a metal pan, add 250 mls of 0.1X SSC and bring to a roaring boil.
> 2) Turn off heat and immediately add 1.25 mls of 20% SDS.
> 3) Immediately add blot and submerge it.
> 4) Place on a rocker platform and rock 10-15 minutes. <--"basting"
> 5) When cool or warm, check with G_counter and and repeat if necessary.
> 6) Image o/n (film or imager) to determine level of previous probe.
> 7) Re-probe!
> With this method, I've often been able to re-probe genomic southerns and
> northerns 3-4 times before having to "retire" the blot.
> In your case, with the blot already dried. You are likely stuck until it
> decays out. There is something about dried blots wherein the probe is
> locked into place and I've never been able to both remove the probe from
> dried blot and still have the blot in good enough shape to be re-probed.
> I've always found those mutually exclusive and often easier just to
> the blot.
> Suggestion: After I'm finished exposing a blot, I always place it at 4'C
> still wrapped in saran wrap. I've placed one of those plastic magazine
> holders in the cold room and just "pack-rat" all my blots in the holders.
> I'd advise folk just to keep them wrapped and cold.
> Hope this helps,
> David L. Haviland, Ph.D.
> Asst. Prof. Immunology
> University of Texas - Houston, H.S.C.
> Institute of Molecular Medicine
> 2121 W. Holcombe Blvd.
> Houston, TX 77030
> Internet:"dhavilan at imm2.imm.uth.tmc.edu"
> Voice: 713.500.2413 FAX: 713.500.2424
> The hardness of butter is directly proportional to the softness
> of the bread.
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