IUBio

DNA Seq core facility set-u

Janice Janice
Thu Oct 3 02:04:55 EST 1996


                       Subject:                               Time:3:14 PM
  OFFICE MEMO          DNA Seq core facility set-up           Date:10/2/96

To all who work in/manage/direct DNA sequencing core facilities:

We are in the process of setting up an automated DNA sequencing core 
facility here at the Salk Institute.  We will be starting out with 2 ABI 
377 machines, and we anticipate that both will be used to capacity right 
away based on survey results.  We have done a limited amount of our own 
sequencing over the last few months with pretty good results, but would 
like to get as much input from other experienced people as possible prior 
to making services available to the Institute as a whole.  

We have so far used all materials and protocols recommended by ABI.  We 
have been able to cut the dye-terminator reaction volume in half, but 
otherwise have not strayed from the protocols.  On a good day, with 48cm 
plates we can get about 700 good bases maximum.  Often the reads are 
significantly shorter.  Therefore, one issue is how to modify the 
protocol to maximize read length.  Any suggestions for changes (maybe 
voltage, acrylamide %, running temp, buffer)?

We are interested to hear how people are dealing with template issues.  
Do you suggest/demand of customers a certain method of template prep for 
good results?  Do you make them responsible for template quantitation and 
quality?  Do you do template preparation for them?

Also, there are apparently many methods of post-reaction purification 
(removal of dye terminators) that work with varying degrees of 
reliability and reproducibility.  We'd be interested to hear 
preferences/recommendations from others with more experience.

We are also considering purchasing robotics, and would like input from 
people currently using either the Qiagen robot or the ABI catalyst.  We 
have had demos of both machines, so we are familiar with their 
capabilities.  What we want to know is how well they work in the field, 
and how useful they really are in a core facility setting.

Lastly, any suggestions regarding data management--both sequence data and 
customer usage/charge-back data--would be well appreciated.

Thanks for your help!!

Janice Wahlstrom
The Salk Institute
Dept MBVL
10010 N. Torrey Pines Rd.
La Jolla, CA  92037
tel:  619-453-4100 x1464
fax:  619-457-4765
wahlstrom at salk.edu





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