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Genomic cloning troubleshooting: Evaluating the success of partial fill-in reactions
with the Xho I half-site arms cloning strategy
Gary Kobs
Promega Corporation
Promega's LambdaGEM®-11 and LambdaGEM-12 vectors are designed to take
advantage of a cloning strategy (1) that relies on the exclusive specificity with which
partially filled-in Xho I arms can be combined with partially filled-in Sau3A
I digested genomic DNA. The only ligation products possible are single copies of genomic
inserts with appropriate arms, since the partial fill-in prevents self-ligation reactions
of vector arms, central stuffer, and genomic fragments. The principal advantages of this
method are that it makes genomic DNA fractionation and dephosphorylation unnecessary, is
very rapid, and requires small amounts of starting material.
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