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Bruce Roe BROE at aardvark.ucs.uoknor.edu
Tue Mar 19 11:34:15 EST 1996

 You write: 
=>    A while back, either in this group or the ABRF mailing list, there was
=> a brief discussion about using DMSO to help sequence through secondary
=> structure problems.  I'm interested in giving my clients in our department
=> the option of adding DMSO to the template/primer mix they submit for
=> automated sequencing (ABI 377, FS, dye-terminator).  I'ld like to hear
=> from people who have used DMSO in this way, in specific the concentration
=> recommended.  Also, since clients would be adding the DMSO to their
=> DNA/primer mix and placing the tube in a fridge where it would remain for
=> up to 72 hours, any information on possible deleterious effects of the
=> DMSO over this kind of period would be appreciated.
=> Eugen Buehler
=> University of Pittsburgh

	With Amplitaq, we have added DISTILLED DMSO to final concentrations
up to 10% and this has improved read-through of difficult areas.  However,
with enzymes containing the Tabor-Richardson mutation, DMSO seems to cause
a loss of enzyme activity in the same concentration range. We have tried
to overcome this by reducing the denaturing temperature from 94degC to
90degC and that seems to help recover some of the lost enzymatic activity.
However, in our hands the lowered temperature plus DISTILLED DMSO with
enzymes containing the Tabor-Richardson mutation, does not seem to give
the improvement in read-through of difficult areas that DMSO does with
Amplitaq. Thus, we are not including DMSO in these reactions and the second
question is a moot point.  For really difficult areas of sequence we have
gone back to Amplitaq and DMSO for another read if the T-R mutation containing
enzyme fails to resolve any ambiguities in the sequence after reading from
both orientations.
	Hope this helps.
Bruce A. Roe, Ph.D    Department of Chemistry and Biochemistry
                      University of Oklahoma, Norman, OK 73019-0370, U.S.A.
Phone: (405) 325-4912 or 7610; FAX: (405) 325-7762; e-mail: broe at uoknor.edu
********************** http://dna1.chem.uoknor.edu/ ***********************

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