Hints for PCR product Autosequencing (ABI)

Luc Simon Luc.Simon at rsvs.ulaval.ca
Tue Dec 20 21:46:58 EST 1994


In article (Dans l'article) <anderson-181294145342 at 128.252.181.14>,
anderson at pharmdec.wustl.edu (Eric C. Anderson) wrote (écrivait) :

> i'm looking for a protocol, or some suggestions regarding sample
> preparation for direct sequencing of PCR products using the ABI Taz Dye
> Deoxy Terminator Sequencing protocol.  I have a few papers that suggest
> using shrimp Alkaline Phosphatase and Exonuclease I, but all the ones that
> i've read deal with radioactive sequencing and Sequenase instead of Taq for
> the polymerase.  anyone with experience doing direct sequencing with the
> ABI kits (either with or without sAP/Exo I) who would like to share hints
> or papers with me would be greatly appreciated.

The suggestions in the ABI manual that should come (you have to ask for
it!) with their Taq DyeDeoxy or Prism ready-made kits are simple,
straightforward, and effective: Purify the excess primers away from your
PCR products (Resins, Ultrafiltration, or precipitation), estimate final
DNA concentration. Then, follow the simple recipe: sequencing reaction mix
+ primer + purified PCR product. Load in the thermal cycler, run it for 25
cycles, purify and load on the automated sequencer.

We usually get very nice reads if proper care is taken in purification
steps and careful estimation of DNA concentration.

-- 
Luc Simon

coordinateur des services scientifiques
recherche en sciences de la vie et de la sante
Universite Laval
Sainte-Foy, Quebec



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