I used Mag-bind total pure NGS beads in 1.2x concentration to clean up my cDNA libraries that are to be used for sequencing. I found that I lost 80% of library during cleanup, is that expected or should I use a lesser concentration of beads during cleanup?
Hi Mansi,
A few things can go wrong when performing a bead cleanup.
Beads tend to settle quickly, it's critical that you ensure that the stock is well mixed before aspirate and dispense into your reaction tubes. I would recommend a really good vortex of the stock (or sub aliquots) before use.
What is the size distribution of your library? Do you perhaps have a fragment distribution trace (labchip/bioanalyzer trace)? I would not use a smaller ratio of beads to reaction product. In general you will exclude smaller fragments when you reduce the amount of beads you add to the cleanup. If you go too low, you will also start excluding some of your library. Having said that, 1.2X is on the high side and you should be capturing most of the smaller fragments as well.
It could be that some of your material is stuck on the beads after the cleanup and elution. Make sure you mix the beads well during the final elution step and allow for a long enough incubation time.
Do you prepare fresh EtOH for use? If your EtOH concentration is out you may be washing away some of you product during the cleanup steps.
Kind regards,
Chris
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