ABI sequencer: reaction clean-up ??
Paul N Hengen
pnh at fcsparc6.ncifcrf.gov
Fri Jan 22 20:19:26 EST 1993
To clean up the Dye-terminator reactions, we use a mini spin-
column exactly as you would for a radiolabelled DNA probe.
There are several versions that have been posted here in the
past for making your own. You should have no problem using them
for the ABI system.
This from Raj Shankarappa (bsh at med.pitt.edu):
We now routinely purify the excess dye from reactions using home (lab)-made
quick spin columns. They may not overcome the tedium but they sure are
cheap. We make a hole in the bottom of 0.5 ml eppendorf using a 26 g needle
and add about 25 ul volume of zirconium glass beads. Then I pour sephadex
G-50 in 0.3M NaAc (5g in 60 ml) to the top of the tube. Stick the small
eppendorf inside a 2 ml tube and spin at about 500 rpm for couple of minutes.
Now the tube is transferred to a new 1.5 ml tube and the sample is added on
top. Now I spin at about 1500 rpm for couple of minutes.
The eluate is precipitated by atleast 3 volumes of isopropanol washed with
70% etoh and resuspended for loading.
Note: We simply dry the void in a roto-vac before loading.
Paul N. Hengen
National Cancer Institute
Frederick Cancer Research and Development Center
Frederick, Maryland 21702-1201 USA
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