formamide

kim at m44.unm.edu kim at m44.unm.edu
Mon Nov 4 14:06:21 EST 1991


Hello group:

I am trying to learn about nucleic acid hybridization on membranes (e.g.
Southern or Northern blotting).  When I read discussions in cookbooks, there is
often a statement to the effect that a higher percentage of Formamide in the
hybridization buffer lowers the melting temperature of hybrids, allowing
hybridizations to be done at lower temperatures.  It seems to be the common
method to use a buffer containing 50% formamide at 42 C.

Why is it that no one seems to use a higher percentage of formamide and an even
lower temperature, such as 37 C?  In catalogs, there is a device available at a
very inflated price called a "Hyb-Aid" oven, consisting of a bottle rotator
inside a controlled temperature oven.  If it is practical to use higher
formamide concentrations to do hybridization reactions at 37 C, blots can be
probed in relatively inexpensive roller bottles on a cell culture roller
apparatus in a warm room.  This would allow one to have the advantages of the
roller-hybridization technology, while using existing equipment.

Are there reasons why this is not done?  

Daniel Kim    KIM at FLOVAX.LANL.GOV



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