Biotinylated DNA/streptavidin conjugation

hoffmanp hoffmanp at tupdean1.med.dal.ca
Thu Mar 11 19:17:46 EST 1999


I have a few questions regarding the above technology:
Please send answers to:            dsalloum at is2.dal.ca

1. Does a biotinylated DNA fragment migrate diferently in Agarose gel
(2%) than a non-biotinylated version.

2. Do you have any suggestion as to why primary PCR using a 5'
biotinylated primer(F6B) with a reverse primer R6 on chromosomal DNA
doesn't give a product of expected size while the non-biotinylated
primer (F6) with R6 gives the right product?

3.Does the fact that I run a secondary PCR on a an amplicon, using the
same primers lead to a slightly longer amplicon for the second reaction.

4. Is a 30% conjugation efficiency(determined by using radioactively
labelled amplicon) considered acceptable for a 5' biotinylated 285 bp
fragment?

5. Is there much concern for running a mobility shift assay using a
biotinylated amplicon? ie. Biotin binding protein etc?

6. Does a biotin cause th eprimer to associate with other regions of the
DNA 'cos when I amplify chromosomal DNA using one biotinylated primer
without it's reverse primer, I get an amplicon of high molecular weight.

I hope to hear some suggestions soon 'cos my thesis defense is on march
25.

Danny Salloum.





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