Stratagene Quick-Hyb

Ed Rybicki ED at micro.uct.ac.za
Mon Oct 12 12:30:36 EST 1992


> Hi Ed:
> You've said that you've used the PEG 6000 instead dextrane sulfate by hybridiza-
> tions with RNA probes - do you have any experiences by using PEG 6000 in
> Northerns? If yes did you incubate by 68 Celsius? I use everytime:
> -1M NaCl
> -10% dextran sulfate
> -50% formamide
> -salmon sperm DNA.
> Could you send me contents of your Hyb-mix?
>                            Best regards
>                                                    Peter Pechan
>                                                    M.P.I., Goettingen-Germany

Sorry to take so long - student prac marking frenzy time here!

Dr Maud Purves - our RNA queen - tells me she uses the Boehringer-Mannheim
Genius kit buffers without modification for Northerns (occasionally
putting in formamide if she feels it necessary).  She has used PEG-6000
for a blot on my recommendation (Note: I have never used it for RNA
except in the presence of formamide and using 32P at 42degC); it was a dot-
blot for a student practical, of plant viral RNA probed with cloned DIG-
labelled DNA.  She shortened hyb time to 2 hr, and got excellent results
with a 68degC hyb with no formamide.

So:  buffers a la B-M Genius kit (including blocking reagents)
     DIG-labelled probe
     no formamide
     DIG-labelled DNA probe (and my student has just showed me a classical
        chemiluminescent Northern done with DIG-labelled RNA, B-M buffers
        and formamide, no PEG, which looked wonderful)
     up to 10% PEG-6000
     68degC, 1-2 hr hyb
     colour or chemiluminescent detection.

Good luck!
 ____________________________________________________________________
| Ed Rybicki, PhD             |    "Now you've got the hang of it    |
| (ed at micro.uct.ac.za)        | There's nothing you can't do with it |
| Dept Microbiology           |        If you're very into it        |
| University of Cape Town     |         You can't go wrong...."      |
| Private Bag, Rondebosch     |                                      |
| 7700, South Africa          |               -Mad John              |
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