Template preparations

Mulligan, John mulligan at darwin.com
Sat Dec 17 14:45:10 EST 1994


Anyone interested in a thread on template preparation for sequencing?

We have been trying the Qiagen 96-well M13 purification plates, and have found
that although the yeild is very low ( <25% of that acheived by quantitative
methods such as phenol extraction ) the quality of the template is good and
consistant.  The cost is high, but balanced by the savings in labor and the
potential for automated handling.

Is anyone using these?  Any experience with yield, or tips for greater recovery?

Is anyone using mag beads in high throughput applications?  Any other solid
phase schemes you are willing to talk about?  

We are using 96-well spin columns to purify PCR products for sequencing (as
described by Francois Iris (in Automated DNA sequencing and Analysis) and Kai
Wang (personal communication).  Yield of DNA is good and reliable, something
aroud 80% of the templates sequence well.

Any other schemes for sequencing PCR templates in use?  What are you success
rates?  Is anyone using the no-purification protocol that Lin Zuo presented at the
last Cold Spring Harbor meeting?  (This scheme may depend on access to the new
mutant Taq that ABI will be relaesing in the near future).  

John Mulligan
Director of Sequencing
Darwin Molecular Corporation



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