PCR product purifn. for sequencing

Scrimshaw, Brian J SCRIMSHAWB%GRIEHFS at LINCOLN.CRI.NZ
Mon Jul 4 04:54:08 EST 1994


Purification of PCR products for sequencing.

I have sequenced lots of PCR products manually in the past, and
have tried lots of different ways of purifying PCR products prior
to sequencing.
My method of choice has been to use promega magic (now wizard)
preps. They are fast real easy and give excellent results for PCR
products in the 300-600bp range.
I am currently trying to purify a 118bp band for sequencing. At
this size the wizard prep has a very poor yield after gel
purification (just done it, zip on the gel). Can someone give me
advice of a good way to purify a band this size? I'm most
interested in the super fast spin it and wait 5 minute type
methods since there's less reagents to make up etc (I make up my
own, and don't want to spend hours trying to purify the stuff)
Has anyone tried spinex tubes from Costar?: Would these be good
for a 118bp bit? how do the tubes work?
What about the amicon micropure things? I'm not real sure how
they work, looks like you stick the gel slice in one end and the
gel stays in the mucropure fitting, the DNA goes thru and sticks
on the filter membrane and the buffer goes right thru, is this
right?
Agarase and a microcon is another way, I've never tried this.
Does this work well?
Can anyone who cleans up these size fragments advise what they
find best?

Many thanks in anticipation

Brian Scrimshaw
Wellington
New Zealand



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