Table 5.

Troubleshooting Guide for preparation and use of cellular reagents.

Problem	Possible Cause	Solution
No / poor protein expression	Degraded inducer	Prepare fresh inducer solution or if appropriate, add measured amount of powder directly to the culture.
	Culture over- or undergrown at the time of induction	Prepare a fresh culture for induction.
	Mutated plasmid or protein expression cassette	Sequence the protein expression plasmid and reconstruct or reacquire correct plasmid. Use fresh transformants for preparing cellular reagents.
	Suboptimal induction conditions	Compare the level of expression of your protein in induced bacteria by comparing with duplicate uninduced cultures using SDS-PAGE analysis. Systematically test the effect of different induction conditions on protein levels. For example, initiate induction at culture densities ranging from A600 values of 0.5 to 1.0, use different amounts of inducer, and vary temperature and duration of induction (3–4 h at 37 °C to overnight at 18–30°C). Repeat SDS-PAGE analysis to assess changes in levels of protein expression. In depth guidance on optimizing heterologous protein expression in E. coli may be found in (Francis & Page, 2010).
Wet cellular reagents after 24 h at 37 °C	Exposure of desiccant to moisture	Use moisture-indicating desiccants, such as indicting Drierite™, to visually assess that the desiccant is dry. Use a new batch of desiccants or regenerate dry desiccants, such as calcium sulfate granules, by spreading them in a single layer and incubating in an oven set at 210° C for 1 hour. Tightly cap the desiccant container in which the cellular reagents are being dried.
	Too much bacterial suspension added to each tube	Do not dry more than 35 μL of bacterial suspension per 0.2-mL tube.
	Insufficient desiccant amount	Do not overload desiccant containers with cellular reagents. For example, place three to four 0.2-mL × 8-tube strips in about 250 g of calcium sulfate desiccant.
	Insufficient opening in the tubes for efficient evaporation	Leave the tube caps entirely open. For capped tubes, make a hole through the cap using an 18-gauge syringe needle.
	Desiccant container left ajar	After placing the cellular reagents inside the desiccant container, tightly close the container lid.
Inactive or poorly active cellular reagents	Poor protein induction during preparation	Remake cellular reagents and check level of induced protein by performing SDS-PAGE gel electrophoresis.
	Too many or too few cellular reagents used in the reaction	Optimize the amount of cellular reagents used in the reaction.
	Nuclease activity in cellular reagents	For thermotolerant and thermostable cellular reagents, prepare them in endonuclease-deficient strains and/or heat-treat hydrated cellular reagents prior to use at 65 °C to 95 °C for 5 to 30 min. For thermolabile cellular reagents, prepare them in endonuclease-deficient strains, such as DH5α.