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Old 02-20-2019, 04:10 AM   #1
adamcarol
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Default Low efficiency in bead-based size selection. Can't get rid of large fragments.

Hi everyone!

We’re performing a bead-based size selection of double digested genomic DNA, targeting fragments between 400bp and 700bp, but we’re having some trouble getting rid of larger fragments. Our size selection protocol employs Serapure beads, prepared following the protocol in Faircloth and Glenn (2011), attached.

To test the efficiency of the Serapure in removing the larger fragments, we size selected DNA ladder using different proportions of Serapure (0,55x and 0,65x of DNA volume). Here is the electrophoresis gel of both the DNA remaining in the supernatant and eluted from the beads. Theoretically, we should see the larger fragments in the beads (marked as 2a, 2b, and 4a, 4b), and only the smaller fragments in the supernatant (marked as 1a, 1b, and 3a, 3b). Unfortunately, not all of the larger fragments seem to be binding to the beads and are still visible in the supernatant (although in a lower concentration, considering that the bands are not that bright).

One of our thoughts is that we’re not using the right proportion of PEG. The amount of PEG8000 used in the Serapure preparation is the same as described in the protocol (9g of PEG8000 and 1mL of Sera-mag SpeedBeads to prepare a 50mL Serapure solution).

I read some very helpful threads about bead-size selection, but still couldn't figure out what seems to be the problem with our experiments.
Any help is very welcome. Many thanks in advance.
-c.

Last edited by adamcarol; 02-25-2019 at 04:35 AM. Reason: missing image
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Old 03-12-2019, 11:06 AM   #2
lydsmith
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Hi adamcarol. I can say that the original formulation of home-made SPRI beads as published in Rohland & Reich 2012 (who Faircloth & Glenn do properly credit in their own protocol) work terribly below a ratio of 0.9X or so. I had a lot of problems getting double-sided selections of 0.55x/0.7x to work properly until I came across this alternative recipe from Philippe Jolivet and Joseph W. Foley:
https://openwetware.org/wiki/SPRI_bead_mix

Their recipe has some differences, but the bead mix components end up being the same as Rohland & Reich except that 1) PEG is increased from 18% to 20% and 2) NaCl is increased from 0.5M to 2.5M. The latter seems to be key since I have made hybrid beads (18% PEG / 2.5M NaCl) which approximate commercial ones best of all.
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Old 03-12-2019, 11:17 AM   #3
lydsmith
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P.S. adamcarol: feel free to follow up here or message me directly for more details. I am happy to help others avoid the same bead struggles I encountered. I came across the Jolivet & Foley protocol through dumb luck while looking for something else, and I came across your question equally randomly while I was searching for another protocol. It could be fate ; )
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Old 03-18-2019, 09:30 PM   #4
lydsmith
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P.P.S. Small clarification. My original message should have read:
Their recipe has some differences, but the bead mix components end up being the same as Rohland & Reich except that 1) PEG is increased from 18% to 20% and 2) NaCl is increased from 1.0M to 2.5M. The latter seems to be key since I have made hybrid beads (18% PEG / 2.5M NaCl) which approximate commercial ones best of all.

Cheers!
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Old 03-31-2019, 03:21 PM   #5
adamcarol
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Hey lydsmith, thanks so much for your reply. I've never come across this alternative recipe, it's pretty helpful!
I found this Sera-Mag User Guide , which features some modifications at the procedures, and got a better result. Still not ideal, but waaaay closer to my target size. I will definitely try the alternative recipe, adding the procedure modifications. Maybe is fate indeed, I'm struggling with this size selection for quite some time.

As soon as I test it I'll give you feedback. Hopefully with a nice gel and bioanalyzer picture with my target size beautifully selected haha

Cheers!
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