Cell Preparation
- Thaw and stage capture and lysis materials
- Thaw one PIP tube per sample and RNase Inhibitor on ice
- Stage at RT Partitioning Reagent, Chemical Lysis Buffer 3
- Stage Combination 1.5 ml/0.5 ml tube stand, P200 and P1000 wide and standard
- Stage one 0.5 ml tube per sample
- Warm Cell Suspension Buffer to 37 C and cell suspension if frozen for 60-90 seconds
- decontaminate cell vial with 70% isopropyl alcohol
- thaw remaining ice at RT 30-60 sec
- Use a wide bore P1000 to gently mix and transfer to 15 ml conical tube
- Slowly add 9 ml warmed thawing media (>30s)
- Centrifuge cells at 200 xg for 5 minutes to pellet
- Aspirate as much supernatant as possible without disturbing pellet
- Add 1 ml prewarmed Cell Suspension Buffer, gently mix with wide bore P1000
- Place remaining Cell Suspension Buffer on ice
- Centrifuge at 200 x g for 3 min
- Aspirate as much supernatant as possible
- Use standard bore P100 to add 200-400 ul cold Cell Suspension Buffer and gently mix to resuspend 10-15 times
- Measure cell count
- Use wide bore P1000 to prepare 1,250 live cells/ul in Cell Suspension Buffer
- Measure cell count, repeat 9 and 10 until cell count reached
- place on ice
Capture and Lysis
- Centrifuge Thawed PIP tubes for ~5 seconds and return to ice
- Preheat Dry Bath with 0.5 ml block