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Old 02-13-2013, 10:35 AM   #12
pmiguel
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Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
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Quote:
Originally Posted by razibus View Post
Thank you for your answer, it makes sense. Do you know that from your own experience or did you read a publication on the subject?
Back in the Sanger Sequencing days we frequently made libraries from sheared BAC DNA. Our yields from the gel were atrocious. We tried a bunch of different methods -- but it came down to anything that was silica based did not want to let go of the DNA if it was above 4 kb or so.

We went to a agarase-based method and that sucked too.

Quote:
Originally Posted by razibus View Post
In the zymo kit, they recommend for larger DNA (>10 kb) to heat the elution buffer up to 60-70C. They claim the kit works from 50 bp to 23 kb. Did you try zymo kit or only Qiagen kit?
Actually, I had not noticed that about the Zymo kit. We just tried it for the first time on some ~10 kb DNA cut out of a gel. this week Actually, I did not do the protocol myself so I don't know if the elution buffer was heated. I hope not, that can have detrimental effects on AT rich fragments.

Anyway, we did get DNA out of the slice, and the yield was pretty good.

--
Phillip
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