Saponin Lysis of RBCs: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
(Initialize)
 
No edit summary
 
(7 intermediate revisions by the same user not shown)
Line 1: Line 1:
==Curators==
==Curators==
Add the primary curators here (including their contact info).  For instance,
Jingyang Chen, Seattle Biomedical Research Institute, 307 Westlake Ave N, Suite 500, Seattle, WA, 98109, USA.  jingyang.chen@sbri.org
 
James Hadfield, CRUK Cambridge Research Institute, Robinson Way, Cambridge CB2 0RE. Tel: +44 (0)1223 404250; Fax: +44 (0)1223 404208; email: jamesdothadfieldatcancerdoyorgdotuk.


''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 [[OpenWetWare:Information management/Protocol curators|discussing the idea of protocol curators]].  Please contribute.''
''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 [[OpenWetWare:Information management/Protocol curators|discussing the idea of protocol curators]].  Please contribute.''


==Abstract==
==Abstract==
Replace this sentence with a brief description of the protocol and its goal.
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.


==Materials==
List everything necessary to perform the protocol here. Include all information about suppliers, ordering details, etc.  Links to the suppliers' page on that material are also appropriate and encouraged. Please be aware that users of this protocol may not be working in the same country as you.
===Reagents===
===Reagents===
Biological resources e.g. cell lines, buffers (link to a method for making them), enzymes, chemicals, kits, etc.
*0.15% Saponin in PBS
*1X PBS
*12 mL of Plasmodium falciparum blood culture at 4% hematocrit
 
===Equipment===
===Equipment===
Any equipment used to perform the protocol (link to a method for using them).
*Refrigerated centrifuge capable of holding 15 mL conical tubes
*Microcentrifuge


==Procedure==
==Procedure==
A step by step guide to the experimental procedure.
#Centrifuge parasite culture at 1400 rpm (~484xg) for 3 minutes.
 
#Aspirate media.
If you find it helpfull you could use the following icons to highlight important parts:
#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.
[[Image:Difficult step.png]] icon to highlight difficult steps
#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.
[[Image:Critical step.png]] warn about critical steps
##Each wash step consists of aspirating the supernatant and resuspending the pellet with new wash buffer, before centrifuging.
 
#Aspirate off last wash.
[[Image:Time required.png]] help people plan how long steps will take
#Store at -20 degrees C until ready to use.
 
[[Image:Pause point.png]] where protocol can be interrupted
 
[[Image:Optional step.png]] steps that can be omitted or included
 
==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 <nowiki>*'''~~~~''':</nowiki> in front) and explained. Links to FAQs/tips provided by other sources such as the manufacturer or other websites would be best made here.<br>
Anecdotal observations that might be of use to others can also be posted here; e.g. 'my cells were still floating'.<br>
 
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==
==Discussion==
You can [[Talk:{{PAGENAME}}|discuss this protocol]].  
You can [[Talk:{{PAGENAME}}|discuss this protocol]].  
Tag this page with categories to allow easier indexing and searching.  See [[Categories]] for information on existing categories.


[[Category:Protocol]]
[[Category:Protocol]]
[[Category:Plasmodium falciparum]]
[[Category:Plasmodium falciparum]]

Latest revision as of 19:43, 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 centrifuge capable of holding 15 mL conical tubes
  • Microcentrifuge

Procedure

  1. Centrifuge parasite culture at 1400 rpm (~484xg) for 3 minutes.
  2. Aspirate media.
  3. Resuspend pellet in 1 ml of 0.15% Saponin (in aliquots in freezer) and transfer to a 1.5 ml eppendorf tube.
  4. Incubate for 5 minutes on ice and vortex each minute.
  5. Spin at 6000 rpm for 3 minutes at 4 degrees C.
  6. Wash with 1 ml 1X PBS (spin at 6000 rpm for 3 minutes) 3 to 4 times.
    1. Each wash step consists of aspirating the supernatant and resuspending the pellet with new wash buffer, before centrifuging.
  7. Aspirate off last wash.
  8. Store at -20 degrees C until ready to use.

Discussion

You can discuss this protocol.

Retrieved from "https://openwetware.org/mediawiki/index.php?title=Saponin_Lysis_of_RBCs&oldid=354716"