Saponin Lysis of RBCs: Difference between revisions
Line 16: | Line 16: | ||
==Procedure== | ==Procedure== | ||
# | #Centrifuge parasite culture at 1400 rpm (~484xg) for 3 minutes. | ||
#Aspirate media. | #Aspirate media. | ||
#Resuspend pellet in 1 ml of 0.15% Saponin (in aliquots in freezer) and transfer to a 1.5 ml eppendorf tube. | #Resuspend pellet in 1 ml of 0.15% Saponin (in aliquots in freezer) and transfer to a 1.5 ml eppendorf tube. |
Revision as of 19:42, 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
- 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.
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'.
It might also be good to add an image to show the workflow and timescales for experiment planning.
Acknowledgments
Acnkowledge any help you had in development, testing, writing this protocol.
References
See OpenWetWare:Biblio for information on how to reference within a wiki.
Specific Protocols
Add links to all the OWW protocols that have been used in making the consensus.
Discussion
You can discuss this protocol.
Tag this page with categories to allow easier indexing and searching. See Categories for information on existing categories.