DNA sequence questions

Harold G. Hills hhills at iastate.edu
Thu Apr 2 11:43:42 EST 1998


Please reply to me and not to the whole group.  I'll summarize as best I =
can.

In the directions for use of various PE Applied Biosystems kits:

You have dried samples ready to sequence.

The directions say:

Resuspend in loading buffer:

Vortex and spin.

I'm curious as to how rapidly the samples dissolve in the formamide and
just how much mixing is necessary.

Do people actually vortex the samples?  What do you do to resuspend your
samples.

I'm also interested in  how people doing 96 well format mix the samples.

Then the directions say to heat for two minutes at 95=B0C for two minutes
then put on ice until ready to load.

Amersham directions say to heat at a lower temperature.

How do you accomplish this step particularly if you are loading from a 96
well format?

Are you putting the samples on ice.

At ISU we are still working with tubes.

After adding loading buffer we spin down the buffer in a microfuge and th=
en
put the tubes in A TOMY MICRO TUBE MIXER for 1-2 minutes.  The mixer hold=
s
36 tubes and has variable speeds.

We then heat at 95=B0C for two minutes and then put the tubes on ice unti=
l
ready to load.   See, we do follow the PE Applied Biosystems directions
sometimes.


When I did manual sequencing, we did not cool on ice.  (Yes, I've sequenc=
ed
about 300-500,000 bases manually.)

Hal



Harold G. Hills, Ph.D. DNA Sequencing Specialist	515 294-9585
1184 Molecular Biology Building			    FAX 515 294-1597
Iowa State University                                   hhills at iastate.ed=
u
Ames, IA 50011-3260
http://www.biotech.iastate.edu/Facilities/DSSF/ABRF   For ABRF 97 DNA
sequencing tutorial.
http://www.biotech.iastate.edu/Facilities/DSSF   For info about facility.





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