Cloning of Lis1 and CKAP5

Jasmin Garcia, Robert Matts

Abstract


Lis1 and CKAP5 are proteins that are both linked to type 1 Lissencephaly. Lis1 is a causative gene for Lissencephaly which causes defects in neural positioning. The regulation of Lis1 is still unknown however. CKAP5 is a microtubule associated protein that is essential for spindle orientation. In order to eventually perform mass spectroscopy experiments to identify any post-translational modifications to the proteins, we had to clone Lis1 and CKAP5. This involved doing a polymerase chain reaction (PCR) amplification, ligation of the PCR fragment into the cloning vector and transformation of the vector into E. coli and plating of the transformed bacteria onto plates for selection of transformed colonies. Transformants were inoculated into liquid media for plasmid preparation, restriction digest of the purified plasmid, and running a gel to screen for inserts. Our results DNA sequencing results verified that we have successfully cloned Lis1. We have also obtained an insert for CKAP5. After we have verified the plasmid carries the insert for CKAP5, we can move on to doing a transfection of cell cultures with Lis1 and CKAP5, performing a cell lysate, running SDS-PAGE, western blotting, and mass spectroscopy experiments to study post-translational modifications of the proteins.

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References


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