Sequencing 16S rRNA genes

Douglas G. Luster luster at ncifcrf.gov
Sun Oct 4 17:36:38 EST 1998


We have been successfully sequencing bacterial 16S sequences using
dRhodamine terminators by the (ABI) book protocols, using 16S PCR products
as the template for sequencing reactions.  We clean up the PCR product with
a single chloroform/phenol extraction and salt/ethanol precip, quantitate,
and sequence 50 ng of product with standard E. coli 16S primers.  This
hadn't worked well with other sequences, e.g. fungal ITS regions, but works
great with the 16S sequences.

Hope this helps,

Doug Luster

>
>Hello all,
>
>     I have a user who has sent me pGEM templates containing 1.4kb inserts
>of 16S rRNA genes. I've attempted to sequence them with both dRhodamine
>terminators and BigDye terminators with 5% dmso, but no luck. Is anyone out
>there sequencing 16S with success, and if so, how are you doing it?
>
>*******************************************
>A Johnson, Ph.D.
>NCSU DNA Sequencing Facility
>Tel: (919)-513-1007
>FAX: (919)-515-7801
>Email: ajohnson at unity.ncsu.edu
>******************************************

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Douglas G. Luster
Plant Physiologist
USDA, Agricultural Research Service
Foreign Disease-Weed Science Research Unit
1301 Ditto Ave.
Ft. Detrick, MD 21702-5023
Voice: 301-619-7344
FAX 301-619-2880
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