Table 3.

Step(s)	Problem	Possible reason	Solution
3	No colonies	Wrong antibiotic	Check if the correct antibiotic has been used (Ampicillin for LwaCas13a plasmid)
3	No colonies	Inefficient heat-shock	Repeat transformation procedure
8	No visible difference between induced and uninduced	Poor expression	Optimize temperature and IPTG induction conditions
18	No specific protein band in final elute	Protein did not bind to beads or precipitated	Check flowthrough for recombinant protein, try re-batch binding of flow-through with more resin
18	Two protein bands or wrong size	Insufficient sumo protease cleavage	Add more SUMO protease, incubate for 4–12 hours, and monitor cleavage by SDS-PAGE
22	No protein peak	Sample did not bind the column	Salt too high, check FPLC waste for protein and change initial buffer A/B mix ratio
23, 27	Slow spin-filter sample concentration	Glycerol sedimentation or protein precipitation	Carefully pipette up and down and centrifuge again
26	No protein peak	Sample lost during application	Check FPLC waste or spin-filter flow-through
41	Low crRNA concentration	Wrong crRNA design	Check crRNA design for T7 promoter sequence
41	Low crRNA concentration	Ethanol concentration in washing solution too low	Prepare fresh 85% EtOH solution
52A-vii, 52B-vii Box 2 - 12	No signal	No amplification during RPA, wrong primer design	Redesign primers
52A-vii, 52B-vii Box 2 - 12	No signal	No amplification during RPA, contaminants in sample	Re-purify or check presence of nucleic acid by other technologies such as PCR
52A-vii, 52B-vii Box 2 - 12	Signal in all samples including negative control	RNase contamination	Incubate reaction without sample, and remove suspected components to determine if signal is reduced
52A-vii, 52B-vii Box 2 - 12	Signal in all samples including negative control	DNA template contamination in reagent	Use new reagents and primer aliquots