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
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