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Old 08-18-2013, 10:41 AM   #1
Noa
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Location: haifa israel

Join Date: Jun 2011
Posts: 62
Default Making library from edge of sheared DNA smear - possible biases?

Hi
We used a Covaris machine to shear BAC DNA that is supposed to go on a 2x250bp MiSeq run. The specifications used were as recommended by Covaris website:

Target BP (peak) 500:
Peak incident power(W) - 175
Duty factor - 5%
Cycle per burst - 200
Treatment time(s) - 35

An inital run of two BACs gave the expected smear around the 500bp size. We then went ahead and prepp'ed 24 BACs and got smears more around the 250bp size.

We would like to know whether it is still OK to use this sheared DNA to make a library for an Illumina run, where the insert is size selected to be 500-700bp. Our main worry is that since we are size selecting the "edge" of the smear and not the peak, we are somehow biasing against certain DNA regions which will then harm our attempts to assemble the BAC in the downstream bioinformatic analyses.

Many thanks
Noa Sher
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