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high thorugput DNA sequencing


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#1 bepi

bepi

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Posted 06 April 2009 - 08:20 AM

Hello

I am currently sequencing 96-well plates of single chain variable fragments. The DNA is prep'd with Qiagen turbo prep systems and sequenced with ABI 3730 sequencer. As of late I have been getting low quality sequencing reads: reads are short (~400bp) when I am used to ~800bp reads, the 'C's have a shouldering artifact, and the Dye blob renders the first 50bp unreadable.

I have varied the concentration of DNA per reaction from 500ng/rxn to 250ng/rxn.
I have tried using 10% DMSO/rxn
I have tried using 1M Betaine/reaction

and the results are the same.
The capillary array has about 400 hours of work on it but I generally get about 600 hours.

Any suggestions on how to improve the quality of my sequence?

Thanks




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