Table 3 Troubleshooting table

10

No colonies occur after transformation

Use of incorrect competent cells

Check you are using the correct competent cells for corresponding plasmids. If using competent cells other than the recommended cells, results are not guaranteed

Use of incorrect concentration of antibiotics

Check the concentration and shelf life of the antibiotics. The antibiotics should not have passed their expiration date

Plasmids are degraded or badly preserved

Prepare new plasmids with concentration between 1 and 10 ng μL⁻¹

24

Incorrect sizes of fragments occur after restriction enzyme digestion

Wrong colonies have been picked up

Discard the colonies and pick up new colonies

Cultures have been grown for too long

Grow and induce cultures as recommended in the Procedure

Potential mutations have been introduced into the plasmids during the PCR or mutagenesis

Pick up new colonies for screening. Grow the culture at 25 or 30 °C for 48 h instead of at 37 °C overnight

33

Plasmid yield from Maxiprep is low

Bacterial cultures are not well prepared

Reprepare the bacterial cultures. Carefully check the LB media, antibodies, preservation conditions of the glycerol stocks and shaker settings. If possible, scale up culture to >1 L

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

Follow the Maxprep procedures carefully

Incomplete digestion by the restriction enzymes

Check the expiration date of the restriction enzymes

68

No full-length SARS-CoV-2 DNA fragments recovered

Ligation has failed

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 appears as a thin band, perform DNA ligation at 16 °C for 18 h

DNA has degrade

Do not store the DNA fragments at −80 °C

Acidified or expired phenol used during the purification.

Use the phenol as recommended. Do not use acidified phenol

89

Low yield of N-gene DNA

PCR of N-gene did not work.

Re-set up the PCR exactly as recommended in the Procedure

Nonspecific amplification has occurred during the PCR

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. There should be only one DNA band after gel electrophoresis

96

RNA recovery is low

Poor quality of DNA template

Reprepare the ligation and purify high-quality DNA

Low input of DNA template

Increase the in vitro transcription time or the amounts of input template

Contamination of residual phenol

Avoid transferring the organic phase during purification

Too much residual ATP carried over from the ligation

Follow the protocol as recommended. Do not use alcohol for precipitation. Good quality of purified DNA (A260/A230>1, A260/280 ~1.8) is needed for success

97

No CPE/virus at given time

Poor RNA quality

Extend monitoring time after electroporation

Only a small amount of full-length RNA transcribed

Increase the amount of RNA transcripts (up to 60 μg) for transfection

Engineered mutations have significantly attenuated viral replication

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% 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 occurred during plasmid propagation in E. coli

Sequence the plasmids from Maxiprep. Use correct ones to recover the virus

Random cell-adaptive mutations occurred after electroporation

Prepare a new batch of RNA transcripts and redo the electroporation. Use the WT strain as control