Table 1 |.

Troubleshooting table

Step	Problem	Possible reason	Solution
20, 49	Gel does not solidify at all or solidifies very slowly	TEMED and/or APS solution is not fresh	Order new TEMED and/or make fresh APS solution
25, 51	Bromophenol blue and xylene cyanol FF dyes migrate together at the gel front	The gel running buffer may be too old to maintain the pH and ionic strength	Make and use fresh gel running buffer
27	DNA oligos run as a smear spanning the partial or the entire lane	The DNA manufacturer sometimes could underestimate the actual amount of oligo provided in the tube. The gel could be overloaded with too much crude DNA	Measure the DNA concentration yourself using a UV-Vis spectrophotometer at 260 nm to correct the amount of unpurified DNA
41	No DNA pellet or a very small DNA pellet is formed	Too little NaCl (Step 36) is added, and/or the 70% ethanol added to the second centrifugation (Step 39) is not cold enough	Make sure to add enough NaCl before centrifugation, and precool 70% ethanol in −20 °C for at least 3 h before using
53	Gel electrophoresis and AFM imaging may show that a specific DNA star structure does not form at all or at a low yield	The number of unpaired Ts used at the inner corners is not sufficient (if the downstream experimental conditions were checked and optimized)	Change the number of the unpaired Ts by adding or removing one T at a time until the formation yield is ~90% or above
	Nondenaturing gel shows partial DNA star products	The component DNA oligos are not pure or start to breakdown, probably due to DNA nuclease contamination	Check the DNA oligos purity and integrity by running them on a denaturing gel as described in Step 25
72	Mica starts moving during an AFM scan	Thin glue from double-sided tape is displaced by buffer used in tapping in buffer AFM mode	Consider using stronger glue/tape to get the mica adhered to the metal plate
73	Dirty AFM image background	Too much APTES residues are left on the mica surface (Step 69)	Stick to the mica activation procedures of using 0.5% (vol/vol) APTES for 1 min
	AFM imaging cannot resolve the hollow structures on DNA star	Too much DNA is loaded on mica surface and the structures are overlaid on each other (Step 70)	Lower the amount of DNA loaded on the mica. Titrate to find the best working concentration
112, 123, 127, 156,163, 164	Wells exhibit mold or bacterial growth	Cells became contaminated upon initial seeding or during infection	Check diluent, virus stocks and overlay medium for any signs of contamination
		Field virus samples were not collected in a sterile manner	Supplement extra antibiotics such as gentamicin or antimicrobial fungizone in the first agar overlay
127, 130	Agar overlay solidifies before use or during use	Agar solution temperature is too low before preparing the overlay	Check if water baths are set to and measuring the correct temperatures
			If large volumes of overlay are required, aliquot the mixture into smaller volumes and keep at 45 °C until immediately before use
128	A large area of cells does not show neutral red stain	This results from a large area of cell death usually because of monolayer drying during infection or incubation. The cell death could also occur if the temperature of agar overlay, especially the first overlay, is too high	Handle a small number of plates at a time and minimize the time when the cell monolayers are without medium. Additionally, make sure to check the temperature of the overlay mixture before adding onto the cell monolayer
130, 131	Cells show very light staining and plaques do not form clear borders	Incorrect amount of neutral red stain was added to the overlay medium	A third overlay containing the correct amount of neutral red can be added; wait for another day for plaques to appear
		Neutral red might precipitate out of the solution	Add the neutral red to freshly made agar before combining the agar with 2× EMEM
		Unhealthy or older cells also take up less stain	Seed fresh cells
131	There are no apparent or visible plaques shown in the wells	Cells may be lysed	Evaluate cells with microscopy to look for neutral red uptake
		Viral stock may have been diluted too much	Test a more concentrated viral stock from Step 90
		Plaques are very small	Increase incubation time (in Step 130)
136	1,000 to 2,000 RU increase is not seen after SPR chip activation by virus particles	Mix of EDC and NHS are not fresh, and/or virus particles are degraded	Make a fresh EDC/NHS solution and thaw a new aliquot of the virus sample to repeat the SPR chip activation process
140	The SPR response of different DNA nanostructures or of the same structure at different concentrations does not correlate	The DNA complexes do not form well (see Troubleshooting advice for Step 53)	Prepare freshly activated SPR chips, and remake DNA complexes (Steps 42–46)