TROUBLESHOOTING

Step	Problem	Possible reason	Possible solution
10	No colonies occur after transformation	Use wrong competent cells. Use wrong antibiotics Plasmids are degraded or badly preserved.	Use the correct competent cells for corresponding plasmids. Other competent cells than the recommended cells used in this protocol are not guaranteed. Check the concentration and shelf life of the antibiotics. The antibiotics should be not expired. Prepare new plasmids with concentration between 1–10 ng/μl.
24	Incorrect sizes of fragments occur after restriction enzyme digestion	Wrong colonies are picked up. Culture are grown too long. Potential mutations are introduced into the plasmids during the PCR or mutagenesis.	Discard the colonies and pick up new colonies as recommended above. Grow and induce cultures as recommended above. Pick up more new colonies for screening. Grow the culture at 25°C or 30°C for 48 h instead of 37°C for overnight.
33	Plasmid yield from maxiprep is low	Bacterial cultures are not well prepared.	Re-prepare the bacterial cultures. Check carefully of the LB media, antibodies, preservation conditions of the glycerol stocks, and shaker settings. If possible, scale up culture to >1 liter.
45	Low DNA recovery after gel extraction	Low quality of the Maxprep plasmids due to bacterial RNA and/or RNA contamination in the plasmid preparation. Incomplete digestion by the restriction enzymes.	Follow the Maxprep procedures. Check the expiration date of the restriction enzymes.
68	No full-length of SARS-CoV-2 DNA fragments recovered	Ligation fails. DNA breaks down. Wrong or expired phenol used during the purification.	Re-set up the ligation. Always run a portion of purified full-length DNA (1 μl) on a gel to check the ligation efficiency. Large blobs of full-length genomic DNA are needed for success. If full-length DNA appeared as a thin band, do DNA ligation at 16°C for 18 h. Do not store the DNA fragments at −80°C. Use the phenol as recommended. Do not use acidified phenol.
89	Low yield of N-gene DNA	PCR of N-gene did not work. Nonspecific amplification occurs during the PCR.	Re-set up the PCR as recommended. Optimize the PCR conditions if needed. Run a portion of PCR product (1 μl) on a gel to check the quality of the PCR product. It should be only one DNA band after gel electrophoresis.
96	RNA recovery is low	Poor quality of DNA template. Low input of DNA template. Contamination of residual phenol. Too much residual ATP carried over from the ligation.	Re-prepare the ligation and purify high quality DNA. Increase the in vitro transcription time or the amounts of input template. Avoid transferring the organic phase during purification. Follow the protocol as recommended. Do not use alcohol for precipitation. Good quality of purified DNA (A260/A230>1, A260/280 around 1.8) is needed for success.
97	No CPE/virus at given time	Poor RNA quality. Low amount of full-length RNA transcribed. Engineered mutations significantly attenuate viral replication.	Extend monitoring time after electroporation. Increase the amount of RNA transcripts (up to 60 μg) for transfection. Use other approaches (RT-PCR or immunofluorescence) to detect the production of viruses.
97	Significant cell death after electroporation	Cells are too overgrown prior to transfection.	Always use fresh cells with 80–90% of confluence prior to electroporation
108	Yield of DNA fragments for sequencing is low	Virus titer is low.	Increase the input of cDNA template for PCR or pool more viruses for RNA extraction.
108	Undesired mutations occur in the viral genome	Mutations occur when plasmid propagation in E. coli. Random cell-adaptive mutations occur after electroporation.	Sequence the plasmids from Maxiprep. Use correct ones to recover the virus. Prepare a new batch of RNA transcripts and redo the electroporation. Use the WT stain as described in this protocol as a control may help figure it out.