ABI sequencer: reaction clean-up ??

Dr. S.A.J.R. Aparicio saparici at crc.ac.uk
Fri Jan 22 10:48:58 EST 1993



In article <1993Jan22.122927.16338 at news.unige.ch>, mike at medsun.unige.ch (michel morris) writes:
> 	We are about to do our first sequencing on an ABI 
> sequencer, using their DyeDeoxy/Taq pol. protocol. 
> 	ABI recommend (insist!) on cleaning up the reactions 
> before loading, preferably with their own spun columns - surprise, 
> surprise.
> 	Is this really necessary? How do the other protocols
> they mention, such as phenol/CHCl3 or isopropanol ppt. compare?
> What is the quickest way to get the reactions onto the machine 
> (while still getting sequence off...)?


Is this part of the FAQ posting, if not perhaps it should be considered for
inclusion.

The vast majority of fluorescent ddNTPs are not incorporated in the PCR products
of cycle sequencing. If they are not removed effectively they form an enormous peak
at the start of a run and cause streak artifacts for several hundred bases afterwards,
thus seriously degrading the quality of the sequence. In our experience if you see even
traces of dye, by eye, then the sequence is usually so poor as to be unusable.

We do not use spin columns any longer - they are a nuisance to prepare and run and it is
often difficult to get consistent results - in our hands. The key here is consistency. There
are many methods which could remove the ddNTPs but the most reliable is one
developed by Andre Rosenthal and Steve Jones in our lab. This has now been fully published
and the reference is:

New Protocols for DNA sequencing with Dye Terminators,
Rosenthal A, Charnock-Jones DS
J. DNA Sequencing and Mapping; 1992; 3 : 61-64

Briefly, the method relies on using Sephadex G50 as a separation matrix but in static
columns of pre-determined size. The problem of flow is overcome by having triton X100
in the mixture (this seems not to affect the sequencing products). The details are int
the paper. I have had a lot of experience with this now and providing care is taken 
over the making up of the sephadex, the method is highly reproducible. The other caveat
is with lambda DNA, where I take 650ul of sephadex slurry rather than 600 - the large
amounts of high MW dna that come through sometimes bring through traces of ddNTPs as
well.


I hope this is of use to someone.

Dr Sam Aparicio
Molecular Genetics Unit
Dept. Medicine
Cambridge UK



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