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Randomize Gene Sequences with New PCR Mutagenesis Kit

Quantitating PCR product yield: PCR products (10 ml) were electrophoresed on 1% agarose gels along side the 1-kb double-stranded DNA standard provided in the kit. PCR product yields were quantified using the Eagle Eye II still video system as described in the manual for the GeneMorph PCR mutagenesis kit.

lacZ mutagenesis assay: A 650-bp product, containing a 300-bp fragment of lacZ, was amplified from a chloramphenicol resistant lacZ/pBC SK+ plasmid construct. The PCR primers were designed to introduce LIC (ligation independent cloning) sequences at the 5 and 3 ends of the insert. The PCR products were gel purified using the StrataPrep gel extraction kit and then cloned into the Affinity LIC vector as described in the cloning kit manual. SoloPack competent cells were transformed and plated on ampicillin/LB plates. lacZ mutants were scored in a color screening assay by plating transformants on Xgal/IPTG/amp plates. Percent lacZ mutants was calculated as the number of colorless colonies (mutants lacking bgalactosidase activity) divided by the total number of colonies plated (blue plus colorless colonies).

DNA sequencing: Mutation frequencies and mutational spectra were analyzed by DNA sequencing. Individual colonies were randomly selected and resuspended in 200 ml of TE. One ml of each suspension was amplified with Herculase enhanced DNA polymerase using a gene-specific primer and a vector-specific primer. PCR products were purified using the StrataPrep PCR purification kit, and DNA sequencing was performed using a nested
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