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Old 06-22-2017, 09:25 PM   #1
ahay08
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Location: Adelaide, South Australia

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Default SMRTbell library prep for BAC DNA to be run on a Sequel

Hi Ė just wondering if anyone has done a SMRTbell library prep with BAC DNA? I have to sequence 2 BACs, both approx. 150 kb and Iím having trouble shearing them. I need to make 20 kb libraries but I only have Covaris g-tubes to use for shearing (no access to a Megaruptor unfortunately). The g-tubes are partially successful but Iím still seeing HMW DNA (supercoiled?) DNA bands on my PFG in addition to the 20-40 kb sheared products. Iíve tried linearising them which has helped for one, but the other one has a largely unknown sequence and I havenít found any restriction enzymes that only cut the vector. Any help would be greatly appreciated, Iím a new Sequel user/library prep technician, and Iím just discovering the intricacies of the various processes!
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Old 06-22-2017, 10:08 PM   #2
nucacidhunter
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Possible solutions:
Type I topoisomarase treatment before shearing
Increasing centrifuge speed for g-tube
Pippin size selection of input sheared DNA

Larger fragments may not be an issue for 20kb libraries as they might be lost during insane number of purifications during library prep and they are less likely to interfere with sequencing.
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Old 06-22-2017, 10:35 PM   #3
ahay08
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Thanks nucacidhunter, I'll check out the topoisomerase solution. I tried increasing the centrifuge speed but the tubes tend to get clogged with the HMW DNA so nothing at all passes through. Size selection is a good idea too - normally I do a Blue Pippin 15 kb cutoff for a 20 kb library which collects everything over 15, but I could try defining the size range and see how that goes, thanks. Yes I know what you mean with the purifications - definitely an insane number! I've tried using Ampure beads on the BAC DNA but the beads just clump and they won't resuspend no matter what I do. thanks for your help!
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