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| Based on protocol from Mycobacterial Protocols. This doesn't give the peptidoglycan-arabinogalactan-mycoli acid structures, though. They're in the pellet at the end. Those are what I actually want.
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| ==Materials==
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| *1L culture of mycobacteria in 7H9
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| *methanol
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| *petroleum ether
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| *chloroform
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| *0.3% w/v aqueous NaCl
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| ===Solutions===
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| * '''S1''': 10:1 methanol:0.3% w/v aqueous NaCl
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| ==Directions==
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| ===Prepare Culture===
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| # Start with 1L of culture (grown overnight). Centrifuge at 750g for 15 minutes. Discard the supernatant.
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| # Resuspend pellet in 20mL of S1 (10:1 methanol:0.3% NaCl). Transfer to a 125mL glass bottle containing a stirbar.
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| ===Extract apolar lipids===
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| # Add 10mL petroleum ether to bottle. Stir vigorously enough to emulsify contents for 15 minutes.
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| # Let bottle sit until layers separate. Transfer upper layer with a pipette to a 125mL glass bottle for apolar lipids.
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| # Repeat these two steps once more, adding to the same apolar lipid bottle.
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| ===Extract polar lipids===
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| # Add 17.3mL of S2 (9:10:3 chloroform:methanol:0.3% NaCl) to bottle. Stir vigorously for 1h.
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| # Transfer contents of bottle to 50mL conical Falcon tube. Centrifuge at 750g for 15 minutes. Transfer the supernatant to another 50mL tube.
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| # Resuspend the pellet in 5.6mL S3 (5:10:4 chloroform:methanol:0.3% NaCl). Transfer back to glass bottle and stir for 30 minutes. Transfer back to 50mL tube and centrifuge at 750g for 15 minutes, and add supernatant to previous 50mL tube.
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| # Add 9.75mL chloroform and 9.75mL 0.3% aqueous NaCl to supernatant tube. Mix for five minutes, centrifuge at 750g for 10 minutes, and transfer lower layer with a pipette to another tube. This is the polar lipid extract.
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