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Old 03-22-2012, 12:29 AM   #1
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Default weird gel-size selection of 454 library

I used a 300ng Gel extraction product (300-1000bp, electrophoresis condition: TAE, 100V*2.5h, 1.5% low melting temperature GTG Seaplaque agarose) of random-PCR products as a initial material for Rapid library preparation according to the official mannual of Roche 454, but the electropherogram of the Agilent 2100 showed that there were too much fragments>1000bp in the library (see Figure. 1), so I used a “500ng” initial amount of the Gel extraction product to prepare the library again, unlike the routine protocol, I did a gel extraction (electrophoresis condition: TAE, 120V*1.0h, 1.5% standard agarose) after the “3.4 adaptor ligation” but omited the subsequent “3.5 small fragment removal” to get the 300-800bp portion of the ligated library, saftly, a single ampure bead processing (50ul extraction+50ul TE+500ul sizing solution) was conducted to removel the small fragements as possible. Then the desired gel extraction library was subjected to the Agilent 2100 bioanalyzer, the corresponding electropherogram was as Figure. 2.

1) why did so much large fragments>1000bp exist in the Figure.1 & 2 ? I am sure the gel cutting was exact and accurate.

2) Could you give some helps on how to accurately remove the large fragments in the library?

Thank you!
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