<table><tr><td>5, 41,107</td><td>Multiple bands are visible on the electrophoresis gel</td><td>Incomplete digestion of the vector</td><td>Perform digestion for a longer period of time or slice a single band of interest from an agarose gel and purify it with a DNA gel extraction kit (e.g. QIAEX II Gel Extraction Kit from Qiagen)</td></tr><tr><td>3, 38, 105</td><td>Multiple bands are visible on the electrophoresis gel</td><td>PCR conditions or primers do not allow specific amplification</td><td>Modify the PCR conditions or slice the band from the gel with the correct size and purify with a DNA gel extraction kit (e.g. QIAEX II Gel Extraction Kit from Qiagen)</td></tr><tr><td>45</td><td>More than two bands are visible on the electrophoresis gel.</td><td>Incomplete digestion of the vector</td><td>Purify the plasmid as described in Step 45 and repeat digestion from Step 44.</td></tr><tr><td>321</td><td>Low transfection efficiency</td><td>i) Cells are not in good enough condition<br/>ii) PolyFect was not homogenized before transfection and its concentration is not sufficient<br/>iii) PolyFect efficiency has decreased due to poor conservation, or expiration date</td><td>i) Make sure to use cells that are healthy, adherent, preferentially with low passage rate.<br/>ii) Make sure to homogenize the PolyFect solution by inverting or gently tapping the tube (no vortexing).<br/>iii) Try a new batch of PolyFect.</td></tr><tr><td>52</td><td>Absence of amplification using primers P1+P3 (Figure 4)</td><td>i) Problems of amplification with Terra PCR<br/>ii) Absence of recombinant viruses in the population</td><td>i) Proceed to troubleshooting for step 100<br/>ii) Repeat transfection. If the lack of recombinant viruses persists, new vectors should be prepared with different homology arms.</td></tr><tr><td>67</td><td>Similar high production of viruses in samples and negative control</td><td>i) Too long incubation time of viruses<br/>ii) Inefficient drug selection</td><td>i) Use low MOI for Step 84<br/>ii) Replace the stock of the selection drug or increase its concentration. Confirm the lack of contamination of wild-type viruses (parental) and negative control by PCR amplification with primers</td></tr></table>
<table><tr><td></td><td></td><td></td><td>annealing on the selection cassette.</td></tr><tr><td>67</td><td>Apparent lack of viruses in samples</td><td>i) Modification introduced is lethal or highly fitness costing<br/>ii) Low inoculum of viruses</td><td>i) Proceed to genotyping (steps 115-120) to determine the presence or absence of recombinant viruses<br/>ii) Use high MOI for Step 84</td></tr><tr><td>74</td><td>Lack of amplification using primers P1+P3 (Figure 4)</td><td>i) Inefficient TERRA amplification<br/>ii) Modification introduced is lethal</td><td>i) Proceed to troubleshooting 118.<br/>ii) New vectors should be prepared with different homology arms in order to exclude dominant negative effects. If still unsuccessful, repeat transfection and selection using trans-complemented amoebas (Steps 120-125). If trans-complementation also fails, RNA silencing would be a more appropriate tool for the study of the gene of interest.</td></tr><tr><td>100</td><td>Lack of PCR amplification or inconclusive results.</td><td>TERRA polymerase is designed to amplify PCR fragments from unpurified templates without the need for extracting genomic DNA, thereby saving time and money. However, the results may not be conclusive, noisy or some PCR product untenable.</td><td>Perform genomic DNA extraction of leftover samples (as described in supplementary protocol 34-49), followed by PCR amplification with Phusion DNA polymerase (ThermoFisher Scientific).</td></tr></table>

1111

1112 **H1 Related links**

1113 **Key references where this protocol has been used:**

1114 Liu, Y. et al. Virus-encoded histone doublets are essential and form nucleosome-like structures. Cell 184,

1115 4237-4250 e4219 (2021). https://doi.org:10.1016/j.cell.2021.06.032
<table><tr><td></td><td></td><td></td><td>annealing on the selection cassette.</td></tr><tr><td>67</td><td>Apparent lack of viruses in samples</td><td>i) Modification introduced is lethal or highly fitness costing<br/>ii) Low inoculum of viruses</td><td>i) Proceed to genotyping (steps 115-120) to determine the presence or absence of recombinant viruses<br/>ii) Use high MOI for Step 84</td></tr><tr><td>74</td><td>Lack of amplification using primers P1+P3 (Figure 4)</td><td>i) Inefficient TERRA amplification<br/>ii) Modification introduced is lethal</td><td>i) Proceed to troubleshooting 118.<br/>ii) New vectors should be prepared with different homology arms in order to exclude dominant negative effects. If still unsuccessful, repeat transfection and selection using trans-complemented amoebas (Steps 120-125). If trans-complementation also fails, RNA silencing would be a more appropriate tool for the study of the gene of interest.</td></tr><tr><td>100</td><td>Lack of PCR amplification or inconclusive results.</td><td>TERRA polymerase is designed to amplify PCR fragments from unpurified templates without the need for extracting genomic DNA, thereby saving time and money. However, the results may not be conclusive, noisy or some PCR product untenable.</td><td>Perform genomic DNA extraction of leftover samples (as described in supplementary protocol 34-49), followed by PCR amplification with Phusion DNA polymerase (ThermoFisher Scientific).</td></tr></table>

1111

1112 **H1 Related links**

1113 **Key references where this protocol has been used:**

1114 Liu, Y. et al. Virus-encoded histone doublets are essential and form nucleosome-like structures. Cell 184,

1115 4237-4250 e4219 (2021). https://doi.org:10.1016/j.cell.2021.06.032
1116 Bisio, H. et al. Evolution of giant pandoravirus revealed by CRISPR/Cas9. *Nat Commun* **14**, 428 (2023).

1117 https://doi.org:10.1038/s41467-023-36145-4

1118 Alempic, J-M. et al. No fitness impact of the knockout of the two main components of mimivirus genomic

1119 fiber and fibril layer. *bioRxiv*, 2023.04.28.538727 (2023). https://doi.org/10.1101/2023.04.28.538727

1120
1116 Bisio, H. et al. Evolution of giant pandoravirus revealed by CRISPR/Cas9. *Nat Commun* **14**, 428 (2023).

1117 https://doi.org:10.1038/s41467-023-36145-4

1118 Alempic, J-M. et al. No fitness impact of the knockout of the two main components of mimivirus genomic

1119 fiber and fibril layer. *bioRxiv*, 2023.04.28.538727 (2023). https://doi.org/10.1101/2023.04.28.538727

1120