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Old 09-24-2014, 06:42 PM   #1
yxl
Junior Member
 
Location: St. Louis

Join Date: Feb 2011
Posts: 4
Default KAPA, dual-SPRI size selection

The KAPA Hyper kit has a protocol for using AMPure beads for size selection (dual-SPRI), but it seems to me that the beadNA ratio is off (or perhaps I am not understanding the concept correctly). The protocol is below (also on p10-11 of the attached protocol pdf); to summarize, size selection of ~250-450bp fragments is performed with these steps:

Part 1:
- start with adapter-ligated library in 50uL volume
- add 30uL of AMPure XP beads
- place on magnet
- keep the 75uL supernatant and dispose beads (beads contain >450bp fragments)

Part 2:
- the to the 75uL supernatant, add 10uL of AMPure XP beads
- place on magnet
- discard supernatant (contains fragments <250bp)
- wash with 80% EtOH twice
- dry beads
- elute DNA

Part 2, as I understand, is the "left side" selection. The addition of 10uL of AMPure beads to 75uL supernatant is a 10/75 = 0.13x bead ratio; would this not lead to a almost-complete loss of material? It seems to me that a more appropriate AMPure bead volume would be 60uL (60/75=0.8) for a 0.8x bead ratio. Perhaps I'm missing something.


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