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Old 05-05-2013, 07:22 AM   #1
yoyoming1001
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Default I sheared DNA in RSB, will it still work?

I usually use 1x TE but made a mistake and used Illumina's RSB instead. First off, will the shearing still work? And second, if I try to ampure cleanup the sample after shearing, will the DNA bind to the beads? Since it's an elution buffer I'm worried that it wouldn't bind to the beads and I would lose all my material. I typically shear in 130ul and add 180ul of ampure and then elute in RSB.

Any advice or thoughts, good or bad, would be helpful. I don't know what to think, but I'm leaning towards it being bad. Not feeling to great.

Last edited by yoyoming1001; 05-05-2013 at 02:18 PM.
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Old 05-05-2013, 12:30 PM   #2
gabrieltw
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The RSB is 10 mMTris-HCl pH 8.5. It should be fine
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Old 05-08-2013, 01:38 PM   #3
JMC
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I've done this as well and didn't see any issues with my final libraries, so you should be fine.
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Old 05-08-2013, 05:10 PM   #4
yoyoming1001
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Thank you both for your replies. The libraries turned out fine in the end, which I'm so relieved about.
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Old 05-09-2013, 05:36 AM   #5
chevrm
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SPRI bead binding/unbinding has more to do with PEG than anything else. An "elution buffer" (water/basic Tris/etc) will elute off of the beads when NOT in the presence of PEG, which is why you resuspend naked beads (and not beads+PEG) in your elution buffer. In your described 1.4X SPRI cleanup, you will have a 180:130 PEG to Diluant so no worries here. Things start getting weird around 0.4X and below.
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