20.109(S13): TA notes for module 2 - Revision history

AgiStachowiak: /* Day 6 */

2013-04-02T20:59:13Z

Day 6

←Older revision		Revision as of 20:59, 2 April 2013
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	***add 5% β-Me at last minute		***add 5% β-Me at last minute
	*Part 3A: protein purification (see also googledoc!), in ice buckets at their benches		*Part 3A: protein purification (see also googledoc!), in ice buckets at their benches
		+	*Nu
	**Note: prepare solutions ~15% in excess of needed volume		**Note: prepare solutions ~15% in excess of needed volume
	**Water, Charge Buffer		**Water, Charge Buffer

AgiStachowiak: /* Day 6 */

2013-04-02T20:50:40Z

Day 6

←Older revision		Revision as of 20:50, 2 April 2013
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	***about 100μL group, three 300 μL to share should be fine		***about 100μL group, three 300 μL to share should be fine
	***add 5% β-Me at last minute		***add 5% β-Me at last minute
-	*Part 3: protein purification (see also googledoc!), in ice buckets at their benches	+	*Part 3A: protein purification (see also googledoc!), in ice buckets at their benches
	**Note: prepare solutions ~15% in excess of needed volume		**Note: prepare solutions ~15% in excess of needed volume
	**Water, Charge Buffer		**Water, Charge Buffer
	**Binding Buffer, Wash Buffer, and Elution Buffer ''with'' protease inhibitors		**Binding Buffer, Wash Buffer, and Elution Buffer ''with'' protease inhibitors
	**Small BSA aliquots ready.		**Small BSA aliquots ready.
		+	*Part 3B: protein purification
	*Part 4: protein concentration, at teaching bench		*Part 4: protein concentration, at teaching bench
	**put out closer to end of lab		**put out closer to end of lab

AgiStachowiak: /* Day 6 */

2013-04-02T20:44:13Z

Day 6

←Older revision		Revision as of 20:44, 2 April 2013
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	'''Materials required:'''		'''Materials required:'''
		+
		+	Stuff requiring advance thought/prep: protein purification buffers, BSA standards

	*Part 1: cell lysis, all up at teaching bench		*Part 1: cell lysis, all up at teaching bench
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	**Water (150 μL aliquots)		**Water (150 μL aliquots)
	**2X sample buffer available in hood		**2X sample buffer available in hood
-	***add 5% β-Me at last minute)	+	***about 100μL group, three 300 μL to share should be fine
-	~~#Protein~~ purification (see googledoc)	+	***add 5% β-Me at last minute
-	#*Note: prepare solutions ~15% in excess of needed volume	+	*Part 3: protein purification (see also googledoc!), in ice buckets at their benches
-	#*Water, Charge Buffer ~~(actually, will probably will buy pre-charged resin this year)~~	+	**Note: prepare solutions ~15% in excess of needed volume
-	#*Binding Buffer, Wash Buffer, and Elution Buffer w/protease inhibitors	+	**Water, Charge Buffer
-	#*Small BSA aliquots ready.	+	**Binding Buffer, Wash Buffer, and Elution Buffer ''with'' protease inhibitors
-	~~#Protein~~ concentration	+	**Small BSA aliquots ready.
-	#*~~Have~~ 5X Coomassie stain from Bio-Rad, water, ~~tubes ready~~	+	*Part 4: protein concentration, at teaching bench
		+	**put out closer to end of lab
		+	**5X Coomassie stain from Bio-Rad out, three 8 mL aliquots to share (each team needs 2.4 mL)
		+	**9.6 mL aliquot of water, one per team (plus an extra)
		+	**BSA standards --

	'''Day of Lab:'''		'''Day of Lab:'''

-	*~~No quiz - a very busy day!~~	+	*Quiz
		+
		+	MOVING ELSEWHERE
	*Transfer gels to fresh water at end of lab and/or next day.		*Transfer gels to fresh water at end of lab and/or next day.
	*Collect all purified protein samples from students and store at 4 °C.		*Collect all purified protein samples from students and store at 4 °C.

AgiStachowiak: /* Day 7 */

2013-04-02T20:35:40Z

Day 7

←Older revision		Revision as of 20:35, 2 April 2013
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	#*Coomassie bottle and 50 mL conical tubes for measuring		#*Coomassie bottle and 50 mL conical tubes for measuring
	#*Distilled water in 1 L bottles		#*Distilled water in 1 L bottles
		+	#SDS-PAGE, in hood
		+	#*Water baths with boiling chips, turn early on in lab
		+	#*Lid locks
		+	#*Waste bottle for stain
	*Quiz (prepared by TA).		*Quiz (prepared by TA).
	*Post data to wiki.		*Post data to wiki.

AgiStachowiak: /* Day 6 */

2013-04-02T20:35:20Z

Day 6

←Older revision		Revision as of 20:35, 2 April 2013
Line 174:		Line 174:
	'''Materials required:'''		'''Materials required:'''

-	*Part 1: cell lysis	+	*Part 1: cell lysis, all up at teaching bench
	**BPER (3 mL aliquots, 30 μL 10% BSA ''and'' 30 μL protease inhibitors)		**BPER (3 mL aliquots, 30 μL 10% BSA ''and'' 30 μL protease inhibitors)
		+	***last-minute prep, as it should be kept at room temp but has enzymes in it
	**Lysis enzyme available on ice up front		**Lysis enzyme available on ice up front
		+	*Part 2: SDS-PAGE advance prep
	**Water (150 μL aliquots)		**Water (150 μL aliquots)
-		+	**2X sample buffer available in hood
-	~~#SDS-PAGE, ready~~ in hood	+	***add 5% β-Me at last minute)
-	#*~~2X sample buffer (~~add 5% β-Me at last minute)	+
-	~~#*Water baths with boiling chips, turn early on in lab~~	+
-	~~#*Lid locks~~	+
-	~~#*Waste bottle for stain~~	+
	#Protein purification (see googledoc)		#Protein purification (see googledoc)
	#*Note: prepare solutions ~15% in excess of needed volume		#*Note: prepare solutions ~15% in excess of needed volume

AgiStachowiak: /* Day 7 */

2013-04-02T20:33:16Z

Day 7

←Older revision		Revision as of 20:33, 2 April 2013
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	'''Day of Lab:'''		'''Day of Lab:'''

		+	# SDS-PAGE, gel bench
		+	#*Polyacrylamide gels (1 per pair).
		+	#*TGS buffer (1 L per box)
		+	#*Staining boxes, couple of spatulas
		+	#*Coomassie bottle and 50 mL conical tubes for measuring
		+	#*Distilled water in 1 L bottles
	*Quiz (prepared by TA).		*Quiz (prepared by TA).
	*Post data to wiki.		*Post data to wiki.

AgiStachowiak: /* Day 6 */

2013-04-02T20:33:05Z

Day 6

←Older revision		Revision as of 20:33, 2 April 2013
Line 174:		Line 174:
	'''Materials required:'''		'''Materials required:'''

-	~~#Cell~~ lysis	+	*Part 1: cell lysis
-	#*BPER (4 mL aliquots, ~~+40~~ μL 10% BSA and inhibitors)	+	**BPER (3 mL aliquots, 30 μL 10% BSA ''and'' 30 μL protease inhibitors)
-	#*Lysis enzyme available on ice up front	+	**Lysis enzyme available on ice up front
-	#*Water (150 μL aliquots)	+	**Water (150 μL aliquots)
-	~~# SDS-PAGE, gel bench~~	+
-	~~#*Polyacrylamide gels (1 per pair).~~	+
-	~~#*TGS buffer (1 L per box)~~	+
-	~~#*Staining boxes, couple of spatulas~~	+
-	~~#*Coomassie bottle and 50 mL conical tubes for measuring~~	+
-	~~#*Distilled water in 1 L bottles~~	+
	#SDS-PAGE, ready in hood		#SDS-PAGE, ready in hood
	#*2X sample buffer (add 5% β-Me at last minute)		#*2X sample buffer (add 5% β-Me at last minute)

AgiStachowiak: /* Day 5 */

2013-04-02T19:45:02Z

Day 5

←Older revision		Revision as of 19:45, 2 April 2013
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	*Ice buckets out, per two teams to share.		*Ice buckets out, per two teams to share.
	*Part 1		*Part 1
-	**Put out LB for OD measurements, 4 mL per group plus a couple extra tubes.	+	**Put out LB for OD measurements, 10 mL per group plus a couple extra tubes.
	**Sub-cultures		**Sub-cultures
	*** Prepare each BL21 mutant candidate, 6 mL per tube.		*** Prepare each BL21 mutant candidate, 6 mL per tube.
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	**Thaw frozen IPTG or prepare fresh (0.1 M stock). IPTG MW = 238.3 g/mol = 0.095 g in 4 mL for 0.1 M. Put out 550 μL per two teams to share.		**Thaw frozen IPTG or prepare fresh (0.1 M stock). IPTG MW = 238.3 g/mol = 0.095 g in 4 mL for 0.1 M. Put out 550 μL per two teams to share.
	*Part 2		*Part 2
		+	**Thaw digests
	**One-half gel per group and ~ 500 mL TAE buffer per gel available.		**One-half gel per group and ~ 500 mL TAE buffer per gel available.
	***T/R: Three 1% gels and one 1.3% gel.		***T/R: Three 1% gels and one 1.3% gel.
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	'''How it went:'''		'''How it went:'''

-	S13	+	*S13
		+	**T/R: Lots of samples growing quite slowly compared to previous years. Should start at 10 am instead of 10:30 am for W/F. Possible reasons: chloramphenicol made very fresh -- maybe lower slightly? Ditch antibiotics altogether?
		+	**W/F:

	S10		S10

AgiStachowiak: /* Day 5 */

2013-04-01T15:47:13Z

Day 5

←Older revision		Revision as of 15:47, 1 April 2013
Line 125:		Line 125:
	'''Materials required:'''		'''Materials required:'''

		+	*Advance prep
		+	**O/N cultures (see sub-culture notes below)
	*Ice buckets out, per two teams to share.		*Ice buckets out, per two teams to share.
	*Part 1		*Part 1
-	**Put out LB for OD measurements, 4 mL per group plus a couple ~~extras~~.	+	**Put out LB for OD measurements, 4 mL per group plus a couple extra tubes.
	**Sub-cultures		**Sub-cultures
	*** Prepare each BL21 mutant candidate, 6 mL per tube.		*** Prepare each BL21 mutant candidate, 6 mL per tube.
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	**Thaw frozen IPTG or prepare fresh (0.1 M stock). IPTG MW = 238.3 g/mol = 0.095 g in 4 mL for 0.1 M. Put out 550 μL per two teams to share.		**Thaw frozen IPTG or prepare fresh (0.1 M stock). IPTG MW = 238.3 g/mol = 0.095 g in 4 mL for 0.1 M. Put out 550 μL per two teams to share.
	*Part 2		*Part 2
-	**One-half gel per group, TAE buffer, 1 Kbp and 100 bp ladders available. Put out sample sign.	+	**One-half gel per group and ~ 500 mL TAE buffer per gel available.
		+	***T/R: Three 1% gels and one 1.3% gel.
		+	***W/F: Ideal is 2.5 1% gels and 1.5 1.3% gels.
		+	**Loading dye out at teaching bench, about 4 aliquots (50 μL size) for those groups that did not yet add it on D4 and/or who forgot to prepare uncut samples.
		+	**Check which groups didn't prepare uncut WT, have miniprep thawed and available in one or a few shared aliquots as needed.
		+	**1 Kbp and 100 bp ladders available. Two 100 μL aliquots of the 1 Kbp should suffice for both days.
		+	** Put out sample sign. at gel bench as reminder.
		+	*Part 4
		+	**Colonies out at teaching bench

	'''Day of Lab (R/F):'''		'''Day of Lab (R/F):'''
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	'''How it went:'''		'''How it went:'''
		+
		+	S13

	S10		S10

AgiStachowiak: /* Day 5 */

2013-03-30T17:53:46Z

Day 5

←Older revision		Revision as of 17:53, 30 March 2013
Line 125:		Line 125:
	'''Materials required:'''		'''Materials required:'''

		+	*Ice buckets out, per two teams to share.
	*Part 1		*Part 1
-	*#Put out LB for OD measurements, 4 mL per group plus a couple extras.	+	**Put out LB for OD measurements, 4 mL per group plus a couple extras.
-	*#Sub-~~culture~~ each ~~DE3/~~mutant, 6 mL per tube.	+	**Sub-cultures
-	#~~Last year, ~1:20 dilution initiated between 10:00 and 10:30 am worked well.~~	+	*** Prepare each BL21 mutant candidate, 6 mL per tube.
-	~~#This means 4 tubes of mutants per pair.~~	+	*** Prepare enough BL21-wild-type and BL21-reference mutants for each pair to have one tube as needed (plus make two extra of WT, and one of each reference mutant).
-	~~#Also sub-culture~~ enough ~~DE3/~~wild-type and ~~DE3/M124S/E67K/T79P~~ for each pair to have one tube as needed (plus make two extra).	+	***In sum, there should be 4 tubes of BL21 per pair.
-	#Thaw frozen IPTG or prepare fresh (0.1 M stock). IPTG MW = 238.3 g/mol = 0.095 g in 4 mL for 0.1 M.	+	***In the past, ~1:20 dilution initiated between 10:00 and 10:30 am usually worked well. M124S grows somewhat more slowly.
-	#One-half gel per group, TAE buffer, 1 Kbp and 100 bp ladders available. Put out sample sign.	+	**Thaw frozen IPTG or prepare fresh (0.1 M stock). IPTG MW = 238.3 g/mol = 0.095 g in 4 mL for 0.1 M. Put out 550 μL per two teams to share.
		+	*Part 2
		+	**One-half gel per group, TAE buffer, 1 Kbp and 100 bp ladders available. Put out sample sign.

	'''Day of Lab (R/F):'''		'''Day of Lab (R/F):'''