Experiments

This page contains only the experiments that were carried out, not those the theoretically would have followed those performed had they been fully successful.

Assembly PCR of Cas13a

Oligonucleotides of the two parts of Cas13a, overlapping by 25 nucleotides, were diluted according to IDT manufacturer instructions. Several assembly PCRs were performed, the first several of which were unsuccessful due to primer degradation. In the PCR producing a somewhat smudged band of successfully assembled Cas13a DnA, the following protocol was used:

11 micrometers of sigma water, 4 microliters 5x green buffer, .5 micrometers dNTP's, 2 microliters Cas13a primer forward, 2 micrometers Cas13a primer reverse, and .5 micrometers of Tax polymerase were mixed thoroughly by vortexing and then by centrifugation. This was run in a standard PCR program with an annealing temperature of 65 degrees Celsius.