Northern blot problem(s)
Malay
curiouser at ccmb.ap.nic.in
Thu Mar 18 15:18:28 EST 1999
::Personally, I don't like to use Denhardt's. I generally get a much better
:result with much lower background using Church buffer. For my Ph.D.
:thesis, I used it to do dozens upon dozens of Northerns with a cDNA probe
:to a low abundance transcription factor using only total, non-poly-A
:selected RNA. Church buffer is a pain in the butt to make up, but I
:usually make 2 L or so at a time, and that lasts me several months. (I
:don't do as many Northerns as I used to do.)
::Here's the recipe:
:: For 500 ml Church buffer
: ------------------------
:BSA (1%-optional) 5 g
:EDTA (1 mM) 1 ml 0.5 M EDTA
:NaPO4 (0.5 M, pH-7.5) 33.5 g Na2PO4.7H20 + 1 ml 85% H3PO4
:SDS 7% 35 g
Here is a good news for you! We have been using a modified Church Buffer for
all our hybridizations, be it Southern, Nothern or even for an oligomer
probe! And the solution is nothing but -
0.5 M Na2HPO4
7% SDS
Nothing else is required!
:Use just like Denhardt's. Wash with various SSC solutions as you normally
:would. Salmon sperm DNA is not necessary, although it can be added if you
:desire. I don't know if it actually works in Church's to reduce background
:since the background is so low to begin with.
Don't add anything with it! It actually increases background!
The beauty of this solution
:is that you don't need to prehybridize for a long time. 15 minutes at 65
:deg C is more than enough. Even better, if you get greater than 50%
:incorporation of 32P into your probe, you don't have to purify the probe
:before use in this solution.
Exactly!
:Hybridization stringency can be increased by making Church buffer with 15%
:formamide and hybridizing at 65 deg C. You can increase stringency still
:further by using Church buffer + 15% formamide made with only 0.2 mM NaPO4
:and hybridizing at 65 deg C.
You can do that with the modified protocol also!
:This solution will precipitate out if the room gets too cool. Just heat it
:to 65 deg C to redissolve.
:
Yep!
Malay
Malay Kumar Basu
Center for Cellular and Molecular Biology
Hyderabad 500007
I N D I A
Fax: (00-91)40-7171195
Phone: (00-91)40-7172241
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The first step to making a dream come true is to wake up
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curiouser at ccmb.ap.ni.in
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