Table 3.

Recipe for TXTL master mix for PAM determination assay for Cas nucleases expressed from pET vectors. The master mix does not include DNA encoding the Cas nuclease and the gRNA. Note that if the PAM of the Cas nuclease of interest is unknown or if no reporter plasmid was constructed during step 2.1.4, then the reporter plasmid does not need to be added. Note depending on how the Cas nucleases are expressed, the IPTG and/or the p70a-T7RNAP may be omitted. *Final concentration accounts for the extra volume from the addition of the DNA constructs encoding the Cas nuclease and the gRNA.

Component	Volume (μl)	Final Conc.*	Stock Conc.
myTXTL extract	75
Randomized PAM library	1	0.5 nM	50 nM
Reporter plasmid from step 2.1.4	1	0.5 nM	50 nM
p70a-T7RNAP	0.5	0.2 nM	40 nM
IPTG	0.5	0.5 mM	100 mM
Chi6 DNA	2	1 μM	50 μM