Hello All,
I'm having a problem with size selection optimization using AMPure XP beads. Basically, I can't get any size selection regardless of volume added to my DNA. I've been using 1ug of 100 bp ladder in 28 uL H2O or Tris buffer and adding 0.7-1.2 volumes beads at .1x increments. The attached (ugly) gel image shows that there is really no size selection going on. Our protocol calls for a 6 minute incubation at RT but mine went a bit over. Still, I would expect to see some signs of size selection. Any ideas?
FYI, for the samples on the left, I used stock beads, while those on the right are 5X diluted in equal volumes of 40%PEG and 5M NaCl. The library prep protocol I use also has a size selection step that uses diluted beads in 1 part 5M NaCl, .6 parts 40% PEG and .4 parts H2O. At those concentrations, I DO get good size selection expected for each volume.
I'm having a problem with size selection optimization using AMPure XP beads. Basically, I can't get any size selection regardless of volume added to my DNA. I've been using 1ug of 100 bp ladder in 28 uL H2O or Tris buffer and adding 0.7-1.2 volumes beads at .1x increments. The attached (ugly) gel image shows that there is really no size selection going on. Our protocol calls for a 6 minute incubation at RT but mine went a bit over. Still, I would expect to see some signs of size selection. Any ideas?
FYI, for the samples on the left, I used stock beads, while those on the right are 5X diluted in equal volumes of 40%PEG and 5M NaCl. The library prep protocol I use also has a size selection step that uses diluted beads in 1 part 5M NaCl, .6 parts 40% PEG and .4 parts H2O. At those concentrations, I DO get good size selection expected for each volume.
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