Difference between revisions of "Madhadron:MycolicAcidMeasurement"

From OpenWetWare
Jump to: navigation, search
(Removing all content from page)
Line 1: Line 1:
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.
*1L culture of mycobacteria in 7H9
*petroleum ether
*0.3% w/v aqueous NaCl
* '''S1''': 10:1 methanol:0.3% w/v aqueous NaCl
===Prepare Culture===
# Start with 1L of culture (grown overnight).  Centrifuge at 750g for 15 minutes.  Discard the supernatant.
# Resuspend pellet in 20mL of S1 (10:1 methanol:0.3% NaCl).  Transfer to a 125mL glass bottle containing a stirbar.
===Extract apolar lipids===
# Add 10mL petroleum ether to bottle.  Stir vigorously enough to emulsify contents for 15 minutes.
# Let bottle sit until layers separate.  Transfer upper layer with a pipette to a 125mL glass bottle for apolar lipids.
# Repeat these two steps once more, adding to the same apolar lipid bottle.
===Extract polar lipids===
# Add 17.3mL of S2 (9:10:3 chloroform:methanol:0.3% NaCl) to bottle.  Stir vigorously for 1h.
# Transfer contents of bottle to 50mL conical Falcon tube.  Centrifuge at 750g for 15 minutes.  Transfer the supernatant to another 50mL tube.
# 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.
# 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.

Latest revision as of 02:15, 19 October 2007