Difference between revisions of "Algal RNA Extraction"
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1.) Take algae in culture medium and pour 25 ml of culture into 50ml-sized screw top, conical tubes (don’t fill, 25ml of culture in each tube). | 1.) Take algae in culture medium and pour 25 ml of culture into 50ml-sized screw top, conical tubes (don’t fill, 25ml of culture in each tube). | ||
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2.) Spin down tubes in centrifuge; 1000rcf/10mins or until all algae are concentrated in a pellet at the bottom of the tube with clear culture medium above. | 2.) Spin down tubes in centrifuge; 1000rcf/10mins or until all algae are concentrated in a pellet at the bottom of the tube with clear culture medium above. | ||
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3.) Remove the culture medium by gently pouring/or pipetting out (decanting). | 3.) Remove the culture medium by gently pouring/or pipetting out (decanting). | ||
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4.) Add RNAlater to bottom of tube, approximately 10x volume of preservative to concentrated algae, 1-1.5ml RNAlater should be sufficient in most cases). | 4.) Add RNAlater to bottom of tube, approximately 10x volume of preservative to concentrated algae, 1-1.5ml RNAlater should be sufficient in most cases). | ||
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5.) Pipette repeatedly to mix algae in RNAlater, until pellet is fully re-suspended in preservative. | 5.) Pipette repeatedly to mix algae in RNAlater, until pellet is fully re-suspended in preservative. | ||
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6.) Remove all material from 50ml conical tube (important as algae get stuck to the bottom but can be scraped with the pipette tip) and transfer to 1.5ml or 2ml Eppendorf mini-centrifuge tubes. | 6.) Remove all material from 50ml conical tube (important as algae get stuck to the bottom but can be scraped with the pipette tip) and transfer to 1.5ml or 2ml Eppendorf mini-centrifuge tubes. | ||
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7.) Spin Eppi’s at max speed in Beckman centrifuge (5,100rpm) for 10mins, remove top clear RNAlater. | 7.) Spin Eppi’s at max speed in Beckman centrifuge (5,100rpm) for 10mins, remove top clear RNAlater. | ||
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8.) Use pestle to grind, and 27-30gauge needle until homogenized (or any other method of tissue homogenization). | 8.) Use pestle to grind, and 27-30gauge needle until homogenized (or any other method of tissue homogenization). | ||
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9.) Spin Eppi’s at max speed in Beckman (5,100rpm) for 2mins, remove top clear RNAlater. Start Clontech NucleoSpin Plant extraction (Clontech XS kit also a good alternative for small amounts of starting material). Follow kit manual provided. Typical yields 1-15ng/µl (60µl total elution volume). | 9.) Spin Eppi’s at max speed in Beckman (5,100rpm) for 2mins, remove top clear RNAlater. Start Clontech NucleoSpin Plant extraction (Clontech XS kit also a good alternative for small amounts of starting material). Follow kit manual provided. Typical yields 1-15ng/µl (60µl total elution volume). | ||
Revision as of 15:29, 18 November 2011
Protocol for obtaining concentrated cells from algal culture
1.) Take algae in culture medium and pour 25 ml of culture into 50ml-sized screw top, conical tubes (don’t fill, 25ml of culture in each tube).
2.) Spin down tubes in centrifuge; 1000rcf/10mins or until all algae are concentrated in a pellet at the bottom of the tube with clear culture medium above.
3.) Remove the culture medium by gently pouring/or pipetting out (decanting).
4.) Add RNAlater to bottom of tube, approximately 10x volume of preservative to concentrated algae, 1-1.5ml RNAlater should be sufficient in most cases).
5.) Pipette repeatedly to mix algae in RNAlater, until pellet is fully re-suspended in preservative.
6.) Remove all material from 50ml conical tube (important as algae get stuck to the bottom but can be scraped with the pipette tip) and transfer to 1.5ml or 2ml Eppendorf mini-centrifuge tubes.
RNA isolation
7.) Spin Eppi’s at max speed in Beckman centrifuge (5,100rpm) for 10mins, remove top clear RNAlater.
8.) Use pestle to grind, and 27-30gauge needle until homogenized (or any other method of tissue homogenization).
9.) Spin Eppi’s at max speed in Beckman (5,100rpm) for 2mins, remove top clear RNAlater. Start Clontech NucleoSpin Plant extraction (Clontech XS kit also a good alternative for small amounts of starting material). Follow kit manual provided. Typical yields 1-15ng/µl (60µl total elution volume).
