Hello all,
I was prepping samples for WGS Using the Kapa HyperPrep workflow. After the final cleanup with 0.8x AMPure SPRI beads, my gel confirmation showed that most of my samples were lost.
I'm trying to mentally troubleshoot what could have gone wrong, but I don't have extensive experience with library prepping to know where to look for the problem.
I washed the samples+beads in 80% EtOH twice for 30s and eluted in 10mM TE+0.05% Tween20 buffer warmed to 55°C. I air dried the beads for 2 minutes between the wash and elution. I incubated in buffer for 3 minutes before removing the beads and used a plate shaker to assist with DNA re-suspension. The cleanup was done with a bead extractor to minimize sample loss.
I redid the cleanup using pre-warmed 10mM Tris-HCl + 0.05% Tween20 instead of TE as the buffer, but the outcome was the same.
Does anyone have suggestions on what could have happened to effect the affinity of the DNA and beads?
Any ideas are welcome.
I was prepping samples for WGS Using the Kapa HyperPrep workflow. After the final cleanup with 0.8x AMPure SPRI beads, my gel confirmation showed that most of my samples were lost.
I'm trying to mentally troubleshoot what could have gone wrong, but I don't have extensive experience with library prepping to know where to look for the problem.
I washed the samples+beads in 80% EtOH twice for 30s and eluted in 10mM TE+0.05% Tween20 buffer warmed to 55°C. I air dried the beads for 2 minutes between the wash and elution. I incubated in buffer for 3 minutes before removing the beads and used a plate shaker to assist with DNA re-suspension. The cleanup was done with a bead extractor to minimize sample loss.
I redid the cleanup using pre-warmed 10mM Tris-HCl + 0.05% Tween20 instead of TE as the buffer, but the outcome was the same.
Does anyone have suggestions on what could have happened to effect the affinity of the DNA and beads?
Any ideas are welcome.
Comment