Is FORMAMIDE necessary for hybridizations?

Alan Smith alansmith at students.wisc.edu
Mon Jan 22 13:16:04 EST 2001


If I remember right formamide greatly increase the amount of time needed for
a hybridization. Especially for RNA. I prefer not to use it because of
expense and toxicity.

See
Nucleic Acid Hybridization: a practical approach Edited by B D Hames and S J
Higgins.
It has a good section on the effects formamide has on hybridization
kinetics.


-----Original Message-----
From: owner-methods at hgmp.mrc.ac.uk
[mailto:owner-methods at hgmp.mrc.ac.uk]On Behalf Of M. Hertel
Sent: Monday, January 22, 2001 11:04 AM
To: methods at hgmp.mrc.ac.uk
Subject: Re: Is FORMAMIDE necessary for hybridizations?


Patrick Lynch wrote:
>
> Always wanted to ask this - is it best to carry out hybridizations in the
> presence or absence of 50% formamide?
>
> P.


Hello Patrick,
Formamid reduces the hybridization temperature, so you can do Your RNA
hybridizations at 42 C.
An other advantage for hybridizing RNA in Formamid is that possible
contamination of RNase would be inactive.
Always deionize Your Formamid for RNA use with an ion exchange resin if
it is not absolutely free from ionic contaminants.
Of course the church buffer works also very well for northerns if you
want to avoid formamid. For RPA we use up to 80% Formamid, here you need
only some ul...
Moritz



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