Low stringency Southern trouble

[martin LEACH]

Hi kelly,

when I do my southerns I wash very slowly...

i.e. first wash is 1xssc/0.1%sds at room temp for 20'

then I check the blot for counts...if still high I move on to

0.5 x ssc/0.1% sds at rt for 20'

check the counts...


then start to throw in some heat

0.2 x ssc/0.1%sds at 50C for 20'

check counts...

if still hot I move onto

0.1xssc/0.1%sds at 60-65C for 20'

checking the counts between washes is critical....you want the b/ground (a
clean piece of the membrane) to go down to low cpm levels without washing off
all your probe

Martin

KELLY THOME (kmorris1 at opal.tufts.edu) wrote:
: I'm hoping someone can help me with remedial Southern analysis technique :-). 
: I'mtrying, without much success, to do low stringency Southerns (ie, hunting for
: family members in one species/looking for homologs in other species).  My hyb
: and washing conditions for high stringency blotting are pretty standard, I
: guess... hyb is 6X SSC/10X Dextran Sulfate/ 0.5% SDS/ 50micrograms/ml ssDNA at
: 65 degrees, washing is 1X SSC/0.5% SDS at 65 degrees.  I tried lowering the
: SDS to 0.1% and hybing at 42, but then I got NO signal, not even the signal I
: see at high stringency.  I'm getting sort of frustrated at blank blots and
: would appreciate advice from someone with experience in this sort of thing. 
: Thanks in advance!
: 		-Kelly

: Kelly Thome			kmorris1 at opal.tufts.edu
: Department of Pathology
: Brigham and Women's Hospital
: Boston, MA 
: 	All thoughts expressed here are my own!

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