1. loxP-PGK-gb2-hygro-loxP: PCR template (50 ng/µl, 20µl)

2. This manual

“loxP-PGK-gb2-hygro-loxP” template is designed to allow hygromycin selection

in prokaryotic and eukaryotic cells.

The loxP-PGK-gb2-hygro-loxP template encodes the hygromycin resistance

a eukaryotic promoter (PGK) for expression in mammalian cells.

The prokaryotic promoter gb2 is a slightly modified version of the Em7

promoter; it mediates higher transcription efficiency than the normally used Tn5

promoter. The promoter of the mouse Phosphoglucokinase gene (PGK) is

used as eukaryotic promoter. A synthetic polyadenylation signal terminates the

hygromycin expression. The cassette is flanked by loxP sites for later excision

by Cre-recombinase.

Using the provided PCR template one can easily create a loxP-PGK-gb2-

hygro-loxP cassette flanked by any restriction sites to clone the cassette into

the vector of choice. The restriction sites can be introduced by adding the

corresponding sequence in the PCR primer. The template can easily be used

to generate targeting constructs mediated by a single Red/ET Recombination

step.

The “loxP-PGK-gb2-hygro-loxP template” is not linear but plasmid based

strains. The PCR product can therefore be used directly for downstream

applications without any further purification.

We recommend growing the recombined cells between 4 and 24h under nonselective

conditions before streaking the culture on plates conditioned with

100 µg/ml hygromycin plus the antibiotics for the selective marker of the target

vector (e.g. 15 µg/ml chloramphenicol for BAC clones).

At least 20 PCR reactions can be performed using 1µl per reaction as template.