Hybridisation conditions for Oligos

LOGAND logand at msdos.montpellier.inra.fr
Tue Jun 7 03:50:13 EST 1994


Dear All

Molecular techniques are new to me and thus I am somewhat bemused when 
confronted by the variety of hybridisation solutions mentioned in the 
literature. 
I imagine this is all down to freedom of choice and personal 
preference/peccadillos, however I have two questions in particular -

1. In 'Current Protocols' one of the solutions suggested for hybridiastion of 
DNA probes to filters from a cDNA library screening is the formamide buffer 
which I believe is commonly used for Northens as well. However, under the 
section for synthetic oligos it only states the use of aqueous solutions (SSC) 
or tetramethyl ammonium hybridisation solutions. I also read that a simple 
phosphate buffer can be used for DNA probes. Furthermore, in Sambrook et al. 
there is no mention of using formamide solution for screening libraries. Since I
am hybridising my degenerate oligo probe to Northens in formamide can I use the 
same conditions to screen a cDNA library or is there a reason for using other 
solutions when sceening libs. and does this change if its a synthetic oligo 
rather than a piece of natural DNA?


2. Concentrations of oligo in the solution. From the two sources mentioned it is
suggested to use at least 0.125 ng of each individual oligo in the mixture/ml in
one or 0.1 to 1 pmol presumably total oligo mix/ml in the other (these are not 
the same) - I guess these change under different conditions i.e. if you have 
dextran sulfate there etc. Can anyone give my a pointer to a concentration to 
use using a formamide/SSPE buffer from Sambrook et al. or do these things vary 
too much for all the formulas in the books to be essentially redundant?

Thanks in advance

David C. Logan
Montpellier
France

Logand at montpellier.inra.fr






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