Sequencing PCR Products

[Michael G. Tencza]

Wasun Chantratita (asmsi002 at CMU.CHIANGMAI.AC.TH) wrote:
> Dear Netters
>       I am now trying to directly sequence PCR-amplified DNAs by cycle
> sequencing...

Wasun

I have had very good success gel purifying 1.1kB PCR products using
SeaPlaque, a low-melt product from FMC, and removing the DNA from the gel
slice using Gene Clean from Bio 101.  I elute the DNA into 20ul dH2O and
use 0.5-1.5ul as template in the DeltaTaq 2.0 system from US Bio.
Sometimes the sequencing fails, but overall I am having about 80-90%
success on the first try. Failures can normally be rescued by dilution of the
template DNA 1:1 with dH2O indicating the presence of an inhibitor or too
high a template concentration.  No matter, the yield of readable
sequence is normally 200-250 base pairs with a single load.  I have just
started sequencing within the last year, but it seems that this system is
bullet proof, even for a novice.

Good luck,

Mike