Biotinylated DNA/streptavidin conjugation for protein purification

hoffmanp hoffmanp at tupdean1.med.dal.ca
Thu Mar 11 19:38:43 EST 1999


Hi!!

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 differently 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 labeled 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 primer 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.





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