TVA Cell Line Electroporation Optimization

Pilot experiment to determine general translation effects and optimal conditions for electroporation of TVA cell line.

  • Renilla luciferase shows cap dependent translation
  • Firefly luciferase shows cap independent translation
  • Used Promega Dual-Luciferase Reporter Assay System and FB12 Luminometer from Berthold

    Protocol

  1. Warm 5 ml media in 6 well plate

  2. Resuspend cells in 400 ul serum free media. Use 3 million cells per condition.

  3. Add 1 ug pRSTF-CVB3 plasmid per 1 million cells.

  4. Electroporate cells in 0.4 cm cuvette electroporator

  5. Slowly add the 400 ul of electroporated cells to the warm 5 ml of media.

  6. Incubate the cells in media for 2 hours to let them recover.

  7. Aliquot 1ml of cell suspension per well and add the drug treatment.

  8. Incubate for 16 hours.

  9. Spin down cells (500 G, 5 min).

  10. Wash with PBS and spin down again.

  11. Resuspend in 25 ul passive lysis buffer (samples at this point can be stored in the -80 for a long period of time).

  12. Lyse by freezing cells in -80 and thawing, 5 minutes each. Do 2 freeze/thaw cycles.

  13. Combine 10 ul lysate and 50 ul LAR2 substrate and measure in luminometer to read Firefly luciferase (cap independent translation).

  14. Immediately add 50 ul Stop and Glow solution and measure in luminometer to read Renilla luciferase (cap dependent translation).

  15. Compare ratio of Renilla to Firefly luciferase for readouts.

TVA Electroporation Optimization

8 conditions, 3 million cells each (24 million total)

Each condition is with or without pRSTF-CVB3 in separate 0.4 cm cuvettes

250 uF 250 V 250 uF 300 V 975 uF 250 V 975 uF 300 V

Results

All luciferase values too low, likely no transformation


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