TABLE 2.

Troubleshooting table.

Step	Problem	Possible reason	Solution
4, 20 and 25	Incomplete restriction digestion: 3 fragments are seen instead of 2	Too much DNA used for the restriction digestion	Dilute the plasmid DNA and reset the digestion reaction or incubate longer times until the digestion is complete
		The purified plasmid DNA contains residual cellular proteins and needs to be cleaned up	Perform additional standard phenol/chloroform extractions followed by ethanol precipitation using salts52 before resetting the digestion
		Enzyme(s) are old and less efficient or inactive	Add more enzymes and incubate for longer duration.
			Purchase new enzymes if inactive
10, 24 and 31	Few or no colonies	Cells used for transformation have lost competence	Change the method of preparation of competent cells and use the original plasmid vector to check the efficiency of the competent cells
		Low transformation efficiency	The time constant must be 4–5 for maximum efficiency
14	The recombinant plasmid contains no insert	A contaminant colony was selected	Discard the negative clone and screen more colonies. If the problem persists, step back to the previous valid clone and restart the experiment from there
	The recombinant plasmid contains an insert with errors in its sequence	Possible mutations occurred during cloning	Discard the clone and restart the cloning from the previous step
	The recombinant plasmid contains an insert of the wrong size	Possible recombination/deletion occurred during cloning due to the repetitive nature of the insert	Discard the clone and restart the cloning from the previous step
36	Low expression efficiency	mRNA secondary structure formation	Optimize spider silk codon usage for E. coli in design avoiding G- and C-rich codons
		Bacterial clone with low expression level	Screen colonies in the presence of IPTG in culture medium. The smallest colonies usually give highest expression level
52	Total protein not binding to the resin	Either the resin is old or the binding buffer may not have been added to the protein extract. The buffer composition and/or pH are wrong	The binding buffer should be at least in 1:1 ratio with the protein extract. Check the buffer composition. Replace the resin if the problem persists
	Protein of interest not binding to the resin	Possible change in the conformation of the protein concealing the His-tag or loading too much of the total silk protein	Analyze the flow-through and wash fractions to see if the protein of interest is lost during binding or washing.
			To avoid conformation problems, engineer a C-terminal His-tag
	No protein elution peak	The concentration of imidazole in the elution buffer is too low	Increase the concentration of imidazole and use gradient elution
	Too many proteins are present in the eluted fraction	Nonspecific elution of proteins	Increase the washing time and/or decrease the elution buffer concentration
	Degradation of products after purification	Possible proteolysis	Maintain low temperature during the entire extraction and purification processes. Add protease inhibitors to the purification buffers
71	No signal on the X-ray film	Poor transfer or no transfer	Stain the gel with Coomassie stain following transfer to determine if transfer is complete.
			Increase or decrease the transfer time. The duration may vary depending on the protein size. Higher molecular weight proteins require longer transfers and low molecular weight proteins require shorter transfer
		No expression	If the protein marker gives a positive signal but the recombinant proteins are not detectable, verify the identity of the recombinant plasmid clone. The frozen stock may have been contaminated
		Antibody or substrate not working	Some of the proteins in the marker are also His-tagged and serve as a positive control. Replace the antibody or substrate if there is no signal from the protein marker
		Insufficient chemiluminescent substrate or incubation time too short	Add more substrate and increase the time of incubation
	High background and low signal	Inadequate blocking or insufficient washing	Change the blocking agent to bovine serum albumin. Block with 5% (wt/vol) milk and wash stringently by increasing the amount of Tween-20 in the 1× PBST
73–75	Clog of PEEK tubing	Presence of insoluble protein in spinning dope	Dissolve the protein in HFIP and vortex for a longer time. You can also crush the protein agglomerate with a pipette tip
		Presence of residual salts in the lyophilized protein due to inefficient dialysis	Increase dialysis time and number of buffer changes
	No silk formation	Low protein concentration in the spinning dope	Make sure to have a 25–30% protein solution for the spinning dope
		Inadequate solvent for spinning dope preparation or for the coagulation bath	Increase or decrease the amount of water added to the spinning dope. If fibers still do not form, try different alcohols or organic solvent (acetone) as coagulants