Sequencing

Jeff Fairman jc_ag at sprynet.com
Tue Nov 16 10:51:06 EST 1999


The first reaction should be with vector specific primers.  Your sequence
should be flanked by t7/t3 or m13 for/rev if it is cloned into the standard
vectors.  The rule of thumb we use in my laboratory is that you redesign
internal primers between 300 to 400 base pairs into the sequence.  This
allows you to still have strong confidence in the sequence that you are
designing your internal primers against.  We use oligo from Molecular
Biology Insights for primer desgin.  It has a nice automatic search function
for sequencing primers which work the majority of the time.

If you do not desire as much confidence in your sequence you can try one
read through from both ends, but I would not recommend it.

Jeff

-----------------------------------------------------------------------
Jeff Fairman, Ph.D.
Senior Scientist, Pharmacogenomics Research
Clingenix, Inc.
871 Industrial Road, Suite J
San Carlos, CA 94070
(650) 598-7645 (office)
(650) 598-7641 (fax)
jfairman at clingenix.com

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Dr Raja Kota wrote in message ...
>Hi Netters,
>
>   Whats the best way to sequence a fragment which is over 1500 bp in
>length and is cloned into a vector.  Any references shall be appreciated.
>
>Thanks in advance,
>
>Raja






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