Grew up 6L worth of cells to purify RiAFP, and purified RiAFP. Pellets were flash frozen in liquid nitrogen and stored at -80 C before use. Purification of RiGFP was used for ice recrystallization studies, rat liver morphology studies, and C. elegans survival studies.
Designed Received primers for PCR amplification of Kanamycin and RiAFP/RiGFP for eventual cross over PCR and integration into the genome.
Conducted sucessful PCR (insert image). PCR purified sucessful PCR products and treated with DpnI for 30 minutes.
Crossover PCR was sucessful (insert image)
Integrated crossover PCR product into genome of EcNR2 strain
Transformed new EcnR2 strain with plasmid containing T7 polymerase and screened for appropriate colonies.
Received 22 degenerate oligonucleotide sequences from Keck and performed MAGE. Cultures were grown up to midlog post electroporation and freeze-thawed approximately 14 times before plating. Suriving colonies were isolated, and we will soon put them through another cycle of MAGE. The process will be repeated.
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