Lidstrom:in vitro Pathway Assay: Crude Extract Prep - Revision history

Janet B. Matsen: /* Ultracentrifuge */

Janet B. Matsen — Mon, 08 Oct 2012 22:13:13 GMT

Ultracentrifuge

←Older revision		Revision as of 22:13, 8 October 2012
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	# Balance tubes to nearest mg. 1 and 3 should match, 2 and 4 should match (for balancing)		# Balance tubes to nearest mg. 1 and 3 should match, 2 and 4 should match (for balancing)
	## Use green test tube cap to hold it up.		## Use green test tube cap to hold it up.
-	## add ice cold buffer to samples you are diluting (not H<sub>2</sub>2O)	+	## add ice cold buffer to samples you are diluting (not H<sub>2</sub>O)
	# Load "mushroom" into machine. Yes, it is surprisingly wobbly when you set it down, but that is really all you do.		# Load "mushroom" into machine. Yes, it is surprisingly wobbly when you set it down, but that is really all you do.
	# Ultracentrifuge 45,000 rpm, 1 hr, 4<sup>o</sup> C. (This will take longer than an hour due to spin up and spin down times)		# Ultracentrifuge 45,000 rpm, 1 hr, 4<sup>o</sup> C. (This will take longer than an hour due to spin up and spin down times)

Janet B. Matsen: /* BCA (protein concentration determination): */

Janet B. Matsen — Fri, 31 Aug 2012 18:47:55 GMT

BCA (protein concentration determination):

←Older revision		Revision as of 18:47, 31 August 2012
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	== BCA (protein concentration determination): ==		== BCA (protein concentration determination): ==
-	# We share a kit with Ceci & Nate; look on their benches for the black/colored box.	+	# We share a kit with Ceci & Nate; look on their benches for the black/colored box. Instructions: [http://www.piercenet.com/instructions/2161296.pdf here]
	# Clean 96-well plates to load samples in are in the right cabinet above the GC		# Clean 96-well plates to load samples in are in the right cabinet above the GC
	## Use the flat-bottomed ones. (? what are the round bottom ones for?)		## Use the flat-bottomed ones. (? what are the round bottom ones for?)

Janet B. Matsen: /* BCA (protein concentration determination): */

Janet B. Matsen — Fri, 31 Aug 2012 18:43:10 GMT

BCA (protein concentration determination):

←Older revision		Revision as of 18:43, 31 August 2012
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	== BCA (protein concentration determination): ==		== BCA (protein concentration determination): ==
		+	# We share a kit with Ceci & Nate; look on their benches for the black/colored box.
	# Clean 96-well plates to load samples in are in the right cabinet above the GC		# Clean 96-well plates to load samples in are in the right cabinet above the GC
	## Use the flat-bottomed ones. (? what are the round bottom ones for?)		## Use the flat-bottomed ones. (? what are the round bottom ones for?)

Janet B. Matsen: /* Do the night before: */

Janet B. Matsen — Fri, 31 Aug 2012 18:42:25 GMT

Do the night before:

←Older revision		Revision as of 18:42, 31 August 2012
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	## 1X without PMSF is for washing the desalting columns. Make a lot! 40+ mL/sample you will desalt.		## 1X without PMSF is for washing the desalting columns. Make a lot! 40+ mL/sample you will desalt.
	## Decide which solution you will use to do BCA BSA protein quantification and make a few mL extra for this (need ~ 1/10 mL per well plus some more)		## Decide which solution you will use to do BCA BSA protein quantification and make a few mL extra for this (need ~ 1/10 mL per well plus some more)
-	# Weigh out a suitable amount of BSA (2 mg/mL) for the BCA assay	+	# Weigh out a suitable amount of BSA (2 mg/mL) for the BCA assay & consider planning your dilutions/spreadsheet if using a program to analyze it.
	# prepare acetonitrile + pivolate: use spreadsheet to calculate the amount.		# prepare acetonitrile + pivolate: use spreadsheet to calculate the amount.

Janet B. Matsen: /* French Press */

Janet B. Matsen — Fri, 31 Aug 2012 18:19:17 GMT

French Press

←Older revision		Revision as of 18:19, 31 August 2012
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	# mark top of tube each time you french press it		# mark top of tube each time you french press it
	# send sample through a 2nd time		# send sample through a 2nd time
-	# clean each cell before switching to another sample	+	# clean each cell before switching to another sample. Don't forget to spray liquid into the sample exit hole
	# alternate between EV & full construct to reduce bias of one cell pulverizing better or something		# alternate between EV & full construct to reduce bias of one cell pulverizing better or something
	# Move to 2 ml centrifuge tubes if centrifuging (2 tubes/sample)		# Move to 2 ml centrifuge tubes if centrifuging (2 tubes/sample)

Janet B. Matsen: /* Ultracentrifuge */

Janet B. Matsen — Fri, 31 Aug 2012 18:16:18 GMT

Ultracentrifuge

←Older revision		Revision as of 18:16, 31 August 2012
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	====Ultracentrifuge====		====Ultracentrifuge====
-	# Move CE to (disposable) polyallomer ultracentrifuge tubes	+	# Move CE to (disposable) polyallomer ultracentrifuge tubes & use sharpie to label which sample is in the tube
-	~~# Use red anodized tube holder~~ & ~~mark what each~~ is ~~with sharpie & tape.~~	+
	# Balance tubes to nearest mg. 1 and 3 should match, 2 and 4 should match (for balancing)		# Balance tubes to nearest mg. 1 and 3 should match, 2 and 4 should match (for balancing)
	## Use green test tube cap to hold it up.		## Use green test tube cap to hold it up.
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	# Ultracentrifuge 45,000 rpm, 1 hr, 4<sup>o</sup> C. (This will take longer than an hour due to spin up and spin down times)		# Ultracentrifuge 45,000 rpm, 1 hr, 4<sup>o</sup> C. (This will take longer than an hour due to spin up and spin down times)
	## Takes ~ 1 hr 20 min with acceleration & deceleration		## Takes ~ 1 hr 20 min with acceleration & deceleration
-	## Light blinks green after you start it: temperature needs to drop before it spins, even though everything is precooled	+	## Light blinks green after you start it: temperature needs to drop before it spins, even though everything is precooled. If you can't get it to start, it is probably because one of the settings (Speed, Time, <sup>o</sup>C) are blinking; press enter to get out of the setting mode, then you can press start.
	# Collect supernatant in 2 fresh 2 mL tubes (per sample)		# Collect supernatant in 2 fresh 2 mL tubes (per sample)
	# Collect up to 2.5 mL if desalting.		# Collect up to 2.5 mL if desalting.

Janet B. Matsen: /* Desalting */

Janet B. Matsen — Fri, 31 Aug 2012 18:13:58 GMT

Desalting

←Older revision		Revision as of 18:13, 31 August 2012
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	## Keep a close eye out so the top surface essentially never runs out of water.		## Keep a close eye out so the top surface essentially never runs out of water.
	# Equilibrate PD-10 desalting column and desalt HSFs two times. Don’t need to wash column as much between 1st and 2nd passes through the column.		# Equilibrate PD-10 desalting column and desalt HSFs two times. Don’t need to wash column as much between 1st and 2nd passes through the column.
		+	## NOTE: as of 8/30/2012 we are no longer desalting twice!
	## Wash ~ 7X with buffer before loading first sample.		## Wash ~ 7X with buffer before loading first sample.
	## Wash with buffer 3 times between re-running the same sample (don't count the volume used to elute your sample)		## Wash with buffer 3 times between re-running the same sample (don't count the volume used to elute your sample)

Janet B. Matsen: /* BCA (protein concentration determination): */

Janet B. Matsen — Fri, 31 Aug 2012 18:06:49 GMT

BCA (protein concentration determination):

←Older revision		Revision as of 18:06, 31 August 2012
Line 135:		Line 135:
	# prepare working reagent (WR)		# prepare working reagent (WR)
	# use multi-channel pipette to distribute the WR into the wells		# use multi-channel pipette to distribute the WR into the wells
-	# incubate at 37<sup>0</sup>C for 30 min	+	# incubate at 37<sup>o</sup>C for 30 min
	## Ceci says < 30 min is ok, but suggested > 30 min is not good. Janet: look this up.		## Ceci says < 30 min is ok, but suggested > 30 min is not good. Janet: look this up.
	# measure with the plate reader at Betsy's bay.		# measure with the plate reader at Betsy's bay.

Janet B. Matsen: /* BCA (protein concentration determination): */

Janet B. Matsen — Fri, 31 Aug 2012 18:06:34 GMT

BCA (protein concentration determination):

←Older revision		Revision as of 18:06, 31 August 2012
Line 135:		Line 135:
	# prepare working reagent (WR)		# prepare working reagent (WR)
	# use multi-channel pipette to distribute the WR into the wells		# use multi-channel pipette to distribute the WR into the wells
-	# incubate at ~~37oC~~ for 30 min	+	# incubate at 37<sup>0</sup>C for 30 min
	## Ceci says < 30 min is ok, but suggested > 30 min is not good. Janet: look this up.		## Ceci says < 30 min is ok, but suggested > 30 min is not good. Janet: look this up.
	# measure with the plate reader at Betsy's bay.		# measure with the plate reader at Betsy's bay.

Janet B. Matsen: /* Crude Extract Preparation */

Janet B. Matsen — Fri, 31 Aug 2012 18:05:05 GMT

Crude Extract Preparation

←Older revision		Revision as of 18:05, 31 August 2012
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	# Cool ultracentrifuge rotor		# Cool ultracentrifuge rotor
	# Defrost cell pellets on ice		# Defrost cell pellets on ice
-	# Prepare protein buffer(~~and~~ equilibration buffer~~, if needed)~~	+	# Prepare protein buffer (5X) & 1X desalting/equilibration buffer
-	# Resuspend cell pellets in 3 mL protein buffer (1x HEPES buffer + PMSF)	+	# Resuspend cell pellets in 2.5 mL protein buffer (WAS 1x HEPES buffer + PMSF)

	====French Press====		====French Press====
-	# metal cell(s) should be cold (~~4oC~~)	+	# metal cell(s) should be cold (4<sup>o</sup>C)
	# use small cell		# use small cell
	## small cell sits on top of a metal cylinder with short pegs that hold it in place		## small cell sits on top of a metal cylinder with short pegs that hold it in place
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	# BEFORE LOADING SAMPLE:		# BEFORE LOADING SAMPLE:
	## bottom plug in, exit nozzle attached (doesn't have to be super tight: after pressure drop), hand-crank valve tight		## bottom plug in, exit nozzle attached (doesn't have to be super tight: after pressure drop), hand-crank valve tight
		+	# swing the arm that holds the cell in place into position
	# pressure = 1,000 PSI (maximum for the small cell)		# pressure = 1,000 PSI (maximum for the small cell)
	# ratio selector = "med" --> pressurizes cell		# ratio selector = "med" --> pressurizes cell
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	# Move to 2 ml centrifuge tubes if centrifuging (2 tubes/sample)		# Move to 2 ml centrifuge tubes if centrifuging (2 tubes/sample)
	## If going straight to ultracentrifugation, add whole extract to ultracentrifuge tubes		## If going straight to ultracentrifugation, add whole extract to ultracentrifuge tubes
-	# Wash cell & parts and put cells back in ~~4oC~~ room	+	# Wash cell & parts and put cells back in 4<sup>o</sup>C room

	====Centrifuge (optional)====		====Centrifuge (optional)====
	# Do this only if you aren't going to ultracentrifuge samples		# Do this only if you aren't going to ultracentrifuge samples
-	# Centrifuge 14,000 rpm , 4 ~~deg~~ C for 20 min	+	# Centrifuge 14,000 rpm , 4<sup>o</sup> C for 20 min
	# Collect supernatant. This is the crude cell extract		# Collect supernatant. This is the crude cell extract
-	# Turn on ultracentrifuge and start cooling to 4 ~~deg~~ C	+	# Turn on ultracentrifuge and start cooling to 4<sup>o</sup> C
	# Wash cell & parts if not done yet		# Wash cell & parts if not done yet

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	# Balance tubes to nearest mg. 1 and 3 should match, 2 and 4 should match (for balancing)		# Balance tubes to nearest mg. 1 and 3 should match, 2 and 4 should match (for balancing)
	## Use green test tube cap to hold it up.		## Use green test tube cap to hold it up.
-	## add ice cold buffer to samples you are diluting (not ~~H2O~~)	+	## add ice cold buffer to samples you are diluting (not H<sub>2</sub>2O)
	# Load "mushroom" into machine. Yes, it is surprisingly wobbly when you set it down, but that is really all you do.		# Load "mushroom" into machine. Yes, it is surprisingly wobbly when you set it down, but that is really all you do.
-	# Ultracentrifuge 45,000 rpm, 1 hr, 4 ~~deg~~ C. (This will take longer than an hour due to spin up and spin down times)	+	# Ultracentrifuge 45,000 rpm, 1 hr, 4<sup>o</sup> C. (This will take longer than an hour due to spin up and spin down times)
	## Takes ~ 1 hr 20 min with acceleration & deceleration		## Takes ~ 1 hr 20 min with acceleration & deceleration
	## Light blinks green after you start it: temperature needs to drop before it spins, even though everything is precooled		## Light blinks green after you start it: temperature needs to drop before it spins, even though everything is precooled
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	====Desalting====		====Desalting====
	([https://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/1314723116657/litdoc52130800BB_20110830191706.pdf manual])		([https://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/1314723116657/litdoc52130800BB_20110830191706.pdf manual])
-	# [http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences-US/products/AlternativeProductStructure_17402/17085101 Desalting columns] are stored with water filling the top, at ~~4oC~~.	+	# [http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences-US/products/AlternativeProductStructure_17402/17085101 Desalting columns] are stored with water filling the top, at 4<sup>o</sup>C.
	##New ones are stored in Ceci's fridge. Old ones are stored in Janet/Amanda's fridges. They are about $200-300/20 columns so we re-use them.		##New ones are stored in Ceci's fridge. Old ones are stored in Janet/Amanda's fridges. They are about $200-300/20 columns so we re-use them.
	# mark tube with date and # of uses		# mark tube with date and # of uses