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| I am trying to construct shotgun library. To do this I isolate DNA from BAC using standard alkaline lysis methodology. I shear DNA using a sonicator. I always check results of sonication on the gel. If it's ok, I run 1 % low melting gel and cut fragment between 2-5 kb. I isolate DNA from the gel using Qiagen kit and do end repair reaction using Epicenter end repair kit. I clean isolated DNA using Qiagen PCR cleaning kit, do the ligation and after that transformation. I get very good efficiency of transformation but my problem is that I get a lot of small inserts, between 300 and 400 bp long (For 32 checked plasmids I've got only 6 inserts above 1kb). Maybe someone had similar problem and can suggest what can I do to decrease number of small inserts. Cheers Krzysztof |
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| insert , library , problem , shotgun , small |
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