TABLE 3.

Troubleshooting table.

Step	Problem	Potential solution (s)
12	No colonies	Try an alternate source of DNA ligase
		Use more DNA ligase
		Include a crowding agent such as polyethylene glycol 400 (PEG400)
		Conduct the ligation reaction at a lower temperature to aid sticky-end annealing
	Equal number of colonies on real and mock Ligation	Extend the CIP treatment
15, 24	Plasmid does not contain insert	Anneal the nucleotides in a slower manner. Turn off the PCR machine after heating to 95 °C and allow it to slowly cool to room temperature
		Extend the CIP treatment
		Increase the ratio of annealed oligonucleotides to digested plasmid
	None of the colonies or plasmid contain insert	Vary the concentration of DMSO
		Use an alternate polymerase
		Add or extend 5' overhangs on the oligonucleotides to increase their affinity toward the template relative to each other
		Use a gradient of annealing temperatures
26A(xv)	Aldehyde-tagged protein primarily in the insoluble fraction	Use a lower concentration of IPTG during induction
		If the nontagged protein is soluble under these expression conditions, try an alternative aldehyde tag
	Aldehyde-tagged protein primarily in the wash fractions	Lower the concentration of imidazole in the lysis and wash buffers
30	Intense signaL at the bottom of the gel complicates detection	Repeat and run the gel longer to eLute excess fluorophore
		Use a desalting spin column to reduce the amount of fluorophore present before SDS-PAGE