dye removal from ABI terminator reactions.

Basavaraju Shankarappa bsh at MED.PITT.EDU
Thu Nov 19 17:26:11 EST 1992


From: scarr at kean.ucs.mun.ca
Subject: Removing dye from 373A reactions

>Has anyone found a cheap, quick, reliable way of removing excess dye
>terminators from a Cycle Sequencing reaction for an ABI373A sequencer?
>We usually use an isopropanol extraction, but this seems to leave
>variable amounts of the dyes behind. The ABI recommended BioSpin columns
>do a better job, but they are costly and the ritual is slow & tedious.
>Anyone had any experience with other commerical columns?
>Steve Carr 

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.

	I intend to have large number of colmumns poured and stored at 4 C.
I think it can be done.  But I havenot done it yet.  This way, all I have 
to do is do a quick spin, exchange the tubes add sample spin and ppt.
I get my zirconium beads from Biospec products PO Box 772 Bartelsville
OK 74005.  (918)-336-3363.  Cat # 11079-101. $20.00 a pound; lasts for a
life-time. 
Raj Shankarappa
bsh at med.pitt.edu
Univ of Pittsburgh




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