FIX-II Phage library construction - Low yield ! Why ?

dboehm dboehm at gwdg.de
Tue Nov 2 03:38:11 EST 1999


Dear helpful scientist

I'm working on the construction of a genomic phage library of a mutant
mouse genome, and I ordered the complete set of FIX-II Partial Fill-in
Kit, Ligation-Kit and Klenow- Fill-in Kit from STRATAGENE.

First I had problems with the positive Partial Fill-in Control, but I
fixed This up.

So, now the yield of clones after packaging is not satisfied ! The Phage
arms disapperd after ligation to partial digested and filled-in DNA, but
I got only 12000 pfu (Packaging control gave 1,25 E9 pfu)

So, the hypothesis arose, that to much genomic DNA (2 micro g in this
ligation) to 1 micro g FIX-II Vector is to much.
Now I ligated 0,4 microg DNA to 1 microg FIX-II in the hope to get more
pfu.

Is there someone with experience with library construction by partial
digestion, partial fill-in technique and ligating to FIX-II ?

Is my hypothesis right ? The information is important because ligation
and packaging in expensive and time consuming.

AND: Is there someone with a technique to amplify  the FIX-II Vector for
further experiments ?

Thank you

Detlef Böhm

Institut for Human Genetics Göttingen
Heinrich-Düker Weg 12
D-37073 Götingen






More information about the Methods mailing list