re hybridization problems

J Preiss--Seq Anal preissj at CLVAX1.CL.MSU.EDU
Wed Jan 27 13:39:00 EST 1993


Hi Chriss

	Well, you did not give very much information in your request for help.
Since I really can't trouble shoot your method very well, I will just give you
mine.  I use 1.5x SSPE, 1.0% SDS, and 0.5% blotto (Carnation instant milk, 
made fresh) for my hybe and prehybe for both northern and southern work.
That's it.  Nothing else.  I used to use sheard DNA, but have since left that
out with no ill effect.  I specifically do not like any of the "hybridization
enhancers" like denharts, formamide, and dextran.  I find all of them to give
background problems in genomic southerns where it counts most.  I hybredize
at 55 to 65 degrees.  Tm is defined by the following formula in this mix:

Tm=16.6(log[Na+])+0.41(%GC)+81.5+500/(probe length bp)

[Na+] = 0.15 for 1x SSC		log 0.15 = -0.824
      = 0.015 for 0.1x SSC	log 0.015 = -1.824

probe length from standard oligonucleotide procedures = about 250 bp 
	(regardless of the template length)

If GC is assumed to be about 50%, then:
	Tm = 86 C	in 1.0x SSC
	   = 70 C 	in 0.1x SSC

	These numbers are for the wash conditions, which I do in 0.1% to 
1.0% SDS and 0.1x SSC to 1.0x SSC at 55 C.  I find that going below 55
gives higher background, while changing salt gives varried stringency 
without background.

	Good Luck and feel free to write back for more info.

		Dr. Leonard N. Bloksberg
		PreissJ at clvax1.cl.msu.edu
		Dept. of Biochemistry
		Michigan State University




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