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how to get the flanking sequences


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#1 ale

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Posted 21 April 2004 - 06:41 AM

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(:wub:) 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

#2 jadefalcon

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Posted 22 April 2004 - 01:19 AM

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

Edited by jadefalcon, 22 April 2004 - 01:19 AM.

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#3 jadefalcon

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Posted 22 April 2004 - 02:41 AM

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)
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#4 ale

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Posted 23 April 2004 - 09:29 AM

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)

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




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