shotgun library - small insert problem

Ned Mantei mantei at cell.biol.ethz.ch
Tue Feb 17 17:16:56 EST 2004


In article <2fae38ba.0402152148.3304b622 at posting.google.com>,
 klubien at sigma.ar.lublin.pl (Krzysztof Lubieniecki) wrote:

>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

The source of the problem is that small fragments are cloned with much 
higher efficiency than large fragments. Generally if you want 2--5 kb, 
you should cut out a region of the gel corresponding to a higher 
molecular weight range, say 3--7 or 4--8 kb to get 2--5 kb. Two rounds 
of gel electrophoresis also could help, or maybe just run the initial 
gel rather far.

Conceivably changing methods would help, although it seems to be more a 
purification problem. DNase I in the presence of manganese cuts both 
strands. The ends can then be cleaned up with Pfu polymerase or T4 DNA 
polymerase. 
See  Nucleic Acids Res.  1981 Jul 10;9(13):3015-27.  Shotgun DNA 
sequencing using cloned DNase I-generated fragments. Anderson S.

While looking for that citation I came across 
Nucleic Acids Research, Vol 24, Issue 20 3879-3886, 1996 Efficient 
random subcloning of DNA sheared in a recirculating point- sink flow 
system.  PJ Oefner, SP Hunicke-Smith, L Chiang, F Dietrich, J Mulligan 
and RW Davis  
 
The second article also briefly discusses advantages and disadvantages 
of various methods.

-- 
Ned Mantei
Department of Cell Biology, Swiss Federal Institute of Technology
CH-8093 Zurich, Switzerland



More information about the Methods mailing list