Saponin Lysis of RBCs: Difference between revisions
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==Procedure== | ==Procedure== | ||
#Pellet 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. | |||
==Critical steps== | ==Critical steps== | ||
Revision as of 19:41, 28 September 2009
Curators
Jingyang Chen, Seattle Biomedical Research Institute, 307 Westlake Ave N, Suite 500, Seattle, WA, 98109, USA. jingyang.chen@sbri.org
Anyone should feel free to add themselves as a curator for this consensus protocol. You do not need to be a curator in order to contribute. The OpenWetWare community is currently discussing the idea of protocol curators. Please contribute.
Abstract
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.
Reagents
- 0.15% Saponin in PBS
- 1X PBS
- 12 mL of Plasmodium falciparum blood culture at 4% hematocrit
Equipment
- Refrigerated microcentrifuge
Procedure
- Pellet 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.
Critical steps
Referenced from the main protocol, a more thorough explanation of particularly important steps in the protocol.
Troubleshooting
Referenced from the main protocol, an explanation of what can cause things to go wrong with the protocol.
Notes
Referenced from the main protocol, any comments about the protocol should be made here; i.e. how it was developed. Any comments added should be signed (by adding *'''~~~~''': in front) and explained. Links to FAQs/tips provided by other sources such as the manufacturer or other websites would be best made here.
Anecdotal observations that might be of use to others can also be posted here; e.g. 'my cells were still floating'.
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Acknowledgments
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References
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Specific Protocols
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Discussion
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