Difference between revisions of "Saponin Lysis of RBCs"
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Revision as of 19:42, 28 September 2009
Jingyang Chen, Seattle Biomedical Research Institute, 307 Westlake Ave N, Suite 500, Seattle, WA, 98109, USA. email@example.com
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Lyse red blood cells while leaving the Plasmodium falciparum parasite intact with it's parasite membrane and parasitophorous vacoule membrane. Typically used right before freezing down parasites for genomic DNA extraction, or for getting rid of hemoglobin right before running a Western Blot on parasite extracts.
- 0.15% Saponin in PBS
- 1X PBS
- 12 mL of Plasmodium falciparum blood culture at 4% hematocrit
- Refrigerated centrifuge capable of holding 15 mL conical tubes
- Centrifuge parasite culture at 1400 rpm (~484xg) for 3 minutes.
- Aspirate media.
- Resuspend pellet in 1 ml of 0.15% Saponin (in aliquots in freezer) and transfer to a 1.5 ml eppendorf tube.
- Incubate for 5 minutes on ice and vortex each minute.
- Spin at 6000 rpm for 3 minutes at 4 degrees C.
- Wash with 1 ml 1X PBS (spin at 6000 rpm for 3 minutes) 3 to 4 times.
- Each wash step consists of aspirating the supernatant and resuspending the pellet with new wash buffer, before centrifuging.
- Aspirate off last wash.
- Store at -20 degrees C until ready to use.
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