Inquiry re: How to strip DIG DNA Probed Nylon Membranes

Sebastian Bunka Sebastian.Bunka at vu-wien.ac.at
Wed Jul 14 05:40:57 EST 1993


> Date:          13 Jul 93 14:13:12 GMT
> To:            "bionet.molbio.methds-reagnts mail newsgroup" <bionet-news at daresbury.ac.uk>
> From:          901106c at axe.acadiau.ca (TIM CHIPMAN)
> Reply-to:      901106c at axe.acadiau.ca (TIM CHIPMAN)
> Subject:       Inquiry re: How to strip DIG DNA Probed Nylon Membranes..?

> I have been attempting to get DIG DNA detection techniques to work for quite
> some time now (Using a kit from Boehringer Mannheim). I have successfully
> managed to probe many membranes and end up with them totally blue (ie: very
> high backgroud levels). I would like to strip the membranes of color and
> probe, for re-use purposes.
>
> A while ago, I posted this question, and someone told me to boil the
> membranes in 0.2x SET, then store them in the freezer. However, I have no
> idea what SET is, nor do I have the address of the person who sent me this
> protocol.
>
> I do have the hot DMF protocol here from the B-M genius guide manual/
> troubleshooting book. However, given how nasty DMF is, and that I can't find
> any around the lab right now, I was hoping that someone could help me out
> with alternate techniques.
>
> Thanks very much!
>
> Tim Chipman
>
The method recommended from the USB Kit (if DNA is on nylon
membranes):

pour boiling 0.1% SDS solution  over blot and agitate 10 min.
repeat with fresh boiling SDS 2 times,
wash briefly in 2x SSC
prehybridize again as before
(alternatively treat with 50% formamide, 2x SSPE at 65 C for one hour)
20x SSPE is 3.0M NaCl, 0.2M NaH2PO4(H2O), 20mM EDTA, pH 7.4

have fun
Sebastian

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|Inst.f.Bakteriologie                                              |
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