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shotgun library - small insert problem

shotgun library - small insert problem - Protocols and Methods Forum

shotgun library - small insert problem - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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Old 02-16-2004, 05:48 AM
Krzysztof Lubieniecki
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Default shotgun library - small insert problem



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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