The current protocol I use didn't give me satisfying result. The protocol using 5% saponin in 1x PBS and the samples incubated in 37 degree Celsius for erythrocyte lysis. 1x PBS was used for washing and Chelex 6% was used to extract DNA (incubated in boiled water).
When I did PCR, it couldn't yield the targeted gene (using primers rPLU1 and rPLU5), only dimers.
Is there anything wrong with the protocol? The only difference I could find compare to other methods is the incubation temperature when the samples were treated using saponin. Mostly, other methods using 4 degree Celsius rather than 37.
Would anyone like to suggest better method?
Thank you
Dear colleague,
For Erythrocyte Lysis
- Cut out a dry blood spot and place it into a microcentrifuge tube (1.5 ml) using a hole puncher. Don’t forget to label the tube, especially when working on multiple samples. For quality control purposes, also include a positive control blood spot.
- Add 1 ml of 0.5% saponin into the microcentrifuge tube containing the blood spot. Close, vortex, and incubate at 4 0C for at least 4 hours, or better yet, overnight. In this step, saponin complexes with cholesterol in the erythrocyte membrane leading to the formation of pores in the membrane and hence hemolysis. Saponin is a commonly used lysis reagent in freeing parasites (for example, malaria parasites) from red blood cells.
- Spin for a few seconds at 4,000 rpm, to remove drops from the inner lid, and aspirate the saponin from the tube using a non-barrier pipette tip attached to a pasteur pipette on a vacuum assembly machine. A plastic pasteur pipette can also be used. Leave the spot in the microcentrifuge tube.
- Then Wash using 1 ml of phosphate buffered saline into the microcentrifuge, close, vortex, and incubate at 4 0C for 20-30 minutes. Incubating overnight will not cause any harm. This step ensures that saponin is removed from the tube.
- Spin for a few seconds at 4,000 rpm, and then remove the PBS, the same way you removed the saponin. Leave the spot in the microcentrifuge tube.
I hope this helps you
thank you very much for the answer. Have you ever done this protocol before? how about the result?
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