llintott at ACS.UCALGARY.CA
Wed Jun 8 12:43:17 EST 1994
> > So how do you get the stringency up? Formamide acts to lower the melting
> temperature of the duplex. I do Northerns at 55C with 50% formamide and get
> good clean bands. Without the formamide I would have thought that there
> would have been a lot of non-specific hybridization. Or are you talking
> about short oligonucleotide probes?
> Dear Warren,
I asume you are reffering to the melting of the DNA duplex? I
denature my probe (which I make by radom priming from a template
of 300 - 1200 bp) by boiling for 5 min and snap cooling on ice.
I then do my hyb at 60 C. The DNA can not reanel without slow
cooling. I get very nice clean bands and no background with this
method. It is my understanding that fomamide is used with RNA
probes in order to allow lowering of the hyb temperature and
prevent formation of hairpin structures. Why this would be more
of a problem with RNA probes I don't know but since I have never
used them I never tried to find out.
Just my totaly uninformed ideas
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