Biotinylated SSR Probe Help

[michael_ohara]

Hello!

I'm trying to probe microsatellites (SSRs) in a mantid (T. sinensis) using two biotinylated oligo sequences [(CA)₁₀ and (GA)₁₀].  I know the concentration the probes need to be, but I don't know the volume.  I've looked through numerous papers and can't find anyone who's reported the volume used.  I'm going to digest 1ug of genomic DNA with RsaI, hybridize the probes, then pull them out with streptavidin magnetic beads.  There's about 5700 Mbp in a diploid cell if that helps.  Any help would be greatly appreciated.  Thank you!

Mike

[CharonY]

It depends on the system you are using for pull-downs. Often, you want to keep volumes small to have a high total concentration of reactants. However, depending on a lot of variables (specificity of the binding, washing protocols, type of beads etc.) the total volume can be adjusted. Often it is non-critical, as long as one does not go dilute the samples too much. The easiest assessment is starting of with the amount of beads required (by looking at binding capacity), wash them according the protocol and add the same volume of sample (while taking care that the final buffer conditions are correct).