Dandekar & Chandler:AHL Extraction

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AHL Signal Extraction

Recommendation: Use only glass tubes to reduce loss of signal during extraction and storage.

If you must use plastic tubes, be sure to use polypropylene (not polystyrene) tubes.

From LB

In a large test tube (18 mm) containing the sample you wish to test:

1. Add 5mL of ethyl acetate and vortex on high for 30 sec.
2. Let the sample sit for 10 min to allow the two phases to separate.
3. After the 10 min rest, pipette off the clear acetate layer only, being careful to not draw up any of the bottom cell debris layer.
4. Deposit the clear acetate layer in a 10mL glass tube with a sealable air tight cap. This type of vial works well. (Fisher 14-962-26F)
5. Repeat steps 1-4 and add acetate supernatant to collection tube from step 4.
6. Place acetate filled tubes into N2 evaporator until all acetate has evaporated off. (Keep on low level to prevent splashing)
7. Suspend dried signal in 100uL of ethyl acetate, screw sealable cap on tightly, wrap in parafilm and place in -20 freezer.

From casein

In a large test tube (18 mm) containing the sample you wish to test:

1. Add 5mL of ethyl acetate AND "a spatula scoopful of NaCl" and vortex on high for 30 sec.
2. Centrifuge at room temp for 10 min to separate phases.
3. Pipette off the clear acetate layer only being careful to not draw up any of the bottom cell debris layer.
4. Deposit the clear acetate layer in a 10mL glass tube with a sealable air tight cap. This type of vial works well. (Fisher 14-962-26F)
5. Repeat steps 1-4 and add acetate supernatant to collection tube from step 4.
6. Place acetate filled tubes into N2 evaporator until all acetate has evaporated off. (Keep on low level to prevent splashing)
7. Suspend dried signal in 100uL of ethyl acetate, screw sealable cap on tightly, wrap in parafilm and place in -20 freezer.