Dandekar & Chandler:AHL Extraction: Difference between revisions
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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: | |||
# Add 5mL of ethyl acetate and vortex on high for 30 sec. | |||
# Let the sample sit for 10 min to allow the two phases to separate. | |||
# 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. | |||
# Deposit the clear acetate layer in a 10mL glass tube with a sealable air tight cap. [https://www.fishersci.com/shop/products/fisherbrand-disposable-borosilicate-glass-tubes-polypropylene-screw-cap-6/p-196656 This type of vial] works well. (Fisher 14-962-26F) | |||
# Repeat steps 1-4 and add acetate supernatant to collection tube from step 4. | |||
# Place acetate filled tubes into N2 evaporator until all acetate has evaporated off. (Keep on low level to prevent splashing) | |||
# 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: | |||
# Add 5mL of ethyl acetate '''AND''' "a spatula scoopful of NaCl" and vortex on high for 30 sec. | |||
# Centrifuge at room temp for 10 min to separate phases. | |||
# Pipette off the clear acetate layer only being careful to not draw up any of the bottom cell debris layer. | |||
# Deposit the clear acetate layer in a 10mL glass tube with a sealable air tight cap. [https://www.fishersci.com/shop/products/fisherbrand-disposable-borosilicate-glass-tubes-polypropylene-screw-cap-6/p-196656 This type of vial] works well. (Fisher 14-962-26F) | |||
# Repeat steps 1-4 and add acetate supernatant to collection tube from step 4. | |||
# Place acetate filled tubes into N2 evaporator until all acetate has evaporated off. (Keep on low level to prevent splashing) | |||
# Suspend dried signal in 100uL of ethyl acetate, screw sealable cap on tightly, wrap in parafilm and place in -20 freezer. | |||
Latest revision as of 12:42, 12 October 2016
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:
- Add 5mL of ethyl acetate and vortex on high for 30 sec.
- Let the sample sit for 10 min to allow the two phases to separate.
- 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.
- 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)
- Repeat steps 1-4 and add acetate supernatant to collection tube from step 4.
- Place acetate filled tubes into N2 evaporator until all acetate has evaporated off. (Keep on low level to prevent splashing)
- 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:
- Add 5mL of ethyl acetate AND "a spatula scoopful of NaCl" and vortex on high for 30 sec.
- Centrifuge at room temp for 10 min to separate phases.
- Pipette off the clear acetate layer only being careful to not draw up any of the bottom cell debris layer.
- 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)
- Repeat steps 1-4 and add acetate supernatant to collection tube from step 4.
- Place acetate filled tubes into N2 evaporator until all acetate has evaporated off. (Keep on low level to prevent splashing)
- Suspend dried signal in 100uL of ethyl acetate, screw sealable cap on tightly, wrap in parafilm and place in -20 freezer.
