3 replies to this topic

[ale]

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

[jadefalcon]

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.

[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)

[ale]

jadefalcon, on Apr 22 2004, 02:41 AM, said:

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