Madhadron:MycolicAcidMeasurement

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Current revision (05:15, 19 October 2007) (view source)
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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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