primer quality for sequencing

Tom Knight tk at pasteur.ai.mit.edu
Mon Oct 16 22:34:21 EST 2000


alexander.dobrovic at adelaide.edu.au (Alexander Dobrovic) writes:

> Is it true that HPLC purified primers give better results when used for
> sequencing?

I suppose in theory they might.  We get sequence results which vary
almost entirely according to the quality and amount of *template* DNA
not the primers.  We routinely make and use primers for both PCR and
sequencing with the following crude but fast and effective protocol on
our 394:

1) LV40 columns and LV40 program, End CESS, DMT off

2) cap vials tightly, heat at 80C for 1 hour in Reactivap (Pierce)

3) remove vials, allow to cool slightly

4) open vials carefully to avoid boiling over

5) heat open vials at 95C with low volume air in Pierce Reactivap until dried
        (ca. 1/2 hour)

6) Dissolve in 1 ml TE

7) Use 0.3ul as a primer in a 20ul sequencing reaction


The quantity of template DNA is important -- too little and you get
low signal; too much and you get very high signal early in the
sequence, and rapid fall off of signal for the distant bases.







More information about the Methods mailing list