how to get the flanking sequences - lost, pls help (Apr/21/2004 )

hi all....and HELP!!!!!
i m trying to get the sequence of flanking regions from a DNA fragment of 900 bp which encodes part of a key enzyme from a bacterial operon.
I would appreciate VERY MUCH() protocols, literature and suggestions about possible approaoches, particularly:
inverse PCR, working from genomic DNA
use of biotinilated outfacing primers to recover specific amplification with streptavidin.
THANK YOUUUUUUU

-ale-

Hi there!

I did something similar a time ago. I used a modified RACE PCR approach:

Single strand PCR outward of your known sequence, add poly-nucleotide to DNA with terminal transferase, do second PCR with nested primer on the known side and use complementary oligo-nuceotide primer on the other side...

Works!

Published in Biotechniques, around 2000?

Mike

-jadefalcon-

the paper i mentioned:

Restriction Cutting Independent Method for Cloning Genomic DNA Segments Outside the Boundaries of Known Sequences

Knut Rudi, Tonje Fossheim and Kjetill S. Jakobsen
University of Oslo, Norway

BioTechniques 27:1170-1177 (December 1999)

-jadefalcon-

QUOTE (jadefalcon @ Apr 22 2004, 02:41 AM)

thank u 4 the advice, i ll give a try and tell u

-ale-