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Old 11-04-2013, 10:32 AM   #1
mgravina
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Location: Boston

Join Date: Dec 2010
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Default Double-SPRI size selection

We are attempting to automate double-SPRI size selection, in which two successive SPRI-bead-based size cuts are used to select DNA approximately between 200 and 450 bp in length. We are having a very hard time getting the reaction to work and would really appreciate some advice.

Design:
Our design adds Agencourt Ampure SPRI beads to the DNA at a volume ratio of 0.55, incubates and separates out the beads, then moves the supernatant from that binding reaction into a second reaction where the ratio is increased to 0.7. After the second incubation, we separate, rinse, and elute from the beads. The first reaction cuts out the material above 450 bp, and the second cuts out the material below 200 bp.
At least in theory. In reality, the amount of size selection we get is minimal, on the order of maybe 10-20% reduction in the high and low bands.
We have experimented heavily and modified the following factors:
  • Robotic vs manual pipetting
  • Different technicians
  • Different samples, including unselected DNA libraries and DNA ladders
  • Different lots of each reagent, including Ampure beads, ethanol and elution buffer
  • Different SPRI : DNA ratios for each cut point
  • Incubation periods at 5, 10, 15 minutes
  • Smaller and larger volumes
  • Different separation magnets

None of these has improved size selection. We've exhausted our own ideas for what could be causing the problem. Has anyone encountered similar difficulties before, or is there an obvious factor here that we're missing?
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Old 11-04-2013, 03:56 PM   #2
Heisman
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Default

What I did back in the day I was working on this was to make my own solution of PEG and NaCl and not use the bead solution. So, I'd put an aliquot of beads on a magnet, discard the entire supernatant, pour in my own PEG/NaCl mixture, and go from there. In theory it shouldn't make a difference but it worked for me. Something to consider trying.
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Old 11-08-2013, 12:49 PM   #3
JeremyDay
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Can you provide volumes of each reagent/sample?
Vol Starting material=
Vol Ampure added 1st sel=
Volume of Ampure the supernatant is added to=

Starting material is only DNA and H2O or TE, correct? If it is from a RXN, there could be PEG in the RXN. You wash 2x with 70/80% EtOH after second selection?

I successfully use Ampure on Biomek FXP for lib prep and get consistent results, so I may be able to help. Heisman is correct that you dont actually need Ampure beads. In fact, you can make homemade beads and/or just the salt/peg solution. However, if Ampure is not working for you, neither will the solutions.
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Old 11-08-2013, 12:50 PM   #4
JeremyDay
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Also, what is the size of DNA for starting material? Genomic? Fragmented?
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