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IN VIVO SYSTEMS > TISSUE CULTURE

> Phoenix Cell Infection

 

Phoenix Cell Infection Protocol

DAY 1

  • Prepare 5µg or 10µg of DNA in 980µl of autoclaved 150 mM NaCl;
  • Add 16.6µL (5µg) or 33.3 µL (10µg) of ExGen transfection reagent (6eq) TO DNA;
  • Vortex immediately for >30secs and let sit at room temperature for 10 minutes;
  • Aspirate old media (DMEM+10% FBS) from Phoenix cells (60% confluent), add 9 mLs fresh media;
  • Add ExGen/DNA mixture to media and swirl gently;
  • Leave overnight at 37°C;

DAY 2

  • Aspirate media, add 5 mL fresh media;
  • Leave overnight at 32°C;

DAY 3

  • Collect viral supernatant and filter through 0.2µm filter;
  • Add new media (normal for parental cells) to viral supernatant (10mL total);
  • Add 10 µL Polybrene (0.8mg/mL in in 150mM NaCl) per mL of supernatant and add to 60% confluent parental cells;
  • Incubate at 32 °C overnight;

DAY 4

  • Change media to regular parenntal media and leave overnight at 37°C (or go straight to selection);

DAY 5

  • Put in selection media and passage normally in selection for one week or until control parental dish is all dead (you may need to split 1 in 2 into selection but keep everything until you see the efficiency).