ABI sequencing kit

Richard R. Hardy hardy at mighty.fccc.edu
Fri Sep 9 13:03:10 EST 1994


In article <1994Sep9.141500.25869 at oxvaxd>, kclark at immsvr.jr2.ox.ac.uk
(Kevin Clark) wrote:

[concerning the PRISM Taq dye terminator cycle sequencing kit from ABI]

> 
> Richard, Which columns do you use and can you post a brief protocol.
> 

Sure:  (summarized from the ABI kit directions):

1.  Mix 9.5 ul of terminator premix, 5 ul DNA template (containing 0.1 ug
PCR product or 0.5 ug ss DNA or 1 ug ds DNA), primer (0.8 pmol for ss, 3.2
pmol for ds), and water to bring final vol to 20 ul.

2.  Cycle: place tubes in cycler pre-heated to 96c; then 96c for 30sec;
50c for 15 sec; 60c for 4min; 25 cycles total; cool to 4c and hold

3.  Apply to Centri-Sep spin column (details on using this column
omitted); collect sample

4.  Dry sample in vacuum centrifuge (w/o heat)

5.  Resuspend/dissolve sample in 4 ul of 5:1 deonized formamide/50mM EDTA,
pH 8.0

6.  Heat to 90c for 2 min, then chill on ice just prior to loading

That's it.  The recommended columns are from Princeton Separation
(908-431-3338).  With "good" template this kit works really well (at least
in our hands).

-- 
R. Hardy
Member, Institute for Cancer Research,
Fox Chase Cancer Center, Philadelphia, PA
(215) 728-2463



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