This page contains only the experiments that were carried out somewhat successfully, 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 for an extension PCR:
11 micrometers of sigma water, 4 microliters 5x green buffer, .5 microliters dNTP's, 2 microliters Cas13a primer forward, 2 microliters Cas13a primer reverse, and .5 microliters 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.
This was followed by an assembly PCR, which utilized a similar procedure, except was done in a larger total volume of 100 microliters.
A 1.5% agarose gel was prepared, using SYBR safe instead of ethidium bromide for safety. The gel was run at 110 V for 45 minutes, at which point it was imaged, revealing the following.