pcr dna purification for sequencing

felipe wettstein felipe.wettstein at ioez.tu-freiberg.de
Fri Aug 4 06:00:45 EST 2000


> how are you?
fine, thank you, i am waiting for: primers, cryotubes, software-licence
and whatever, but the weather is fine again and tomorow i probably go to
berlin, beautiful nice berlin.

> what is the best method of purification of pcr products for sequencing?
> could you perhaps sketch the protocol briefly?
i would not purify the pcr product, since in your pcr reaction mix there
are only things that you need also for sequencing-pcr, like taq,
template and nucleotides. in our MWG manual for the licor sequencer
there is a sequencing reaction described as follows:

eintrag ins logbuch, MI-3.5.00:
the master mix for one probe
	1.0ul   pcr reaction
	0.4ul	labeled primer (5pmol/ul: total 2pmol (one color))
	7.2ul	sequencing buffer (MWG: Sequitherm Excel II, long read L-C-Kit)
	1.0ul	polymerase EXCEL II
       10.4ul	h2o
 --------------------
       20.0ul	total volume

in every tube i did
	3.5ul mix and 2ul nucleotides (A,C,G,T from the kit)

-------| * ~. -felipe-wettstein----------------------------------------






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