Oneill Lab:Chemicals - Revision history

Seth Kasowitz: /* Blotting Solutions */

Seth Kasowitz — Wed, 17 Oct 2007 14:04:00 GMT

Blotting Solutions

←Older revision		Revision as of 14:04, 17 October 2007
Line 211:		Line 211:

	''Note: SDS will precipitate at low temperatures. We have found keeping Church's Solution at 37°C may help in limiting instances of blot disease''		''Note: SDS will precipitate at low temperatures. We have found keeping Church's Solution at 37°C may help in limiting instances of blot disease''
		+
		+	===5x Random Priming Buffer===
		+	====Components====
		+	*250 mM Tris pH 8.0
		+	*25 mM MgCl<sub>2</sub>
		+	*100 mM NaCl
		+	*10 mM DTT
		+	*1M HEPES
		+	**Adjust pH to 6.6 with high concentration NaOH
		+
		+	====Preparation====
		+	*1.25 mL 1M Tris pH 8.0
		+	*62.5 μL 2M MgCl<sub>2</sub>
		+	*100 μL 5M NaCl
		+	*50 μL 1M DTT
		+	*2.5 mL 2M HEPES
		+	*Bring volume to 5mL with diH<sub>2</sub>O

	===20x Running Buffer===		===20x Running Buffer===

Seth Kasowitz — Fri, 25 May 2007 18:48:03 GMT

Common Lab Chemical Recipes

←Older revision		Revision as of 18:48, 25 May 2007
Line 92:		Line 92:
	*Add 32g NaOH / 2L		*Add 32g NaOH / 2L
	**NaOH cannot be stored in glass containers		**NaOH cannot be stored in glass containers
		+
		+	===ReagentB===
		+	*TEN
		+	**0.1M NaCl
		+	**10mM Tris pH 8.0
		+	**1mM EDTA pH 8.0
		+	*1% SDS
		+	Store at room temperature

	===X-Gal===		===X-Gal===

Seth Kasowitz — Mon, 21 May 2007 14:21:21 GMT

20x Running Buffer

←Older revision		Revision as of 14:21, 21 May 2007
Line 212:		Line 212:
	#Adjust pH to 7.0 with 10N NaOH		#Adjust pH to 7.0 with 10N NaOH
	#Adjust volume to 2L		#Adjust volume to 2L
-	#~~Autoclave~~	+	#Filter sterilize
		+	''This buffer is light sensitive. Store at room temp in a foil wrapped or otherwise light shielded container.''

	==Links and Contacts==		==Links and Contacts==

Seth Kasowitz — Mon, 30 Apr 2007 20:09:32 GMT

←Older revision		Revision as of 20:09, 30 April 2007
Line 213:		Line 213:
	#Adjust volume to 2L		#Adjust volume to 2L
	#Autoclave		#Autoclave
		+
		+	==Links and Contacts==
		+	[[User:Seth\|Seth Kasowitz]] [mailto:seth.kasowitz@uconn.edu]
		+
		+	[http://www.oneill.mcb.uconn.edu/Michael/MONEILLhome.html\|O'Neill Lab Homepage]

Seth Kasowitz — Mon, 30 Apr 2007 20:05:30 GMT

Common Lab Chemical Recipes

←Older revision		Revision as of 20:05, 30 April 2007
Line 28:		Line 28:
	*57.1mL Glacial Acetic Acid		*57.1mL Glacial Acetic Acid
	*Bring final volume to 1L with diH<sub>2</sub>O		*Bring final volume to 1L with diH<sub>2</sub>O
		+
		+	===Column Buffer===
		+	For amylose resin affinity column
		+	*20mL 1.0M Tris pH 7.4
		+	*11.7g NaCl
		+	*2.0mL 0.5M EDTA
		+	*1.0mL 1M NaN<sub>3</sub>
		+	**Very Toxic. Wear gloves and appropriate safety clothing.
		+
		+	===10x Tris-Glycine PAGE Buffer===
		+	For one-dimensional denaturing gels
		+	*15.1g Tris
		+	*94g Glycine
		+	*25ml 20% SDS
		+	*Adjust volume to 500 mL with water
		+	*Store at 4°C
		+
		+	===Isopropanol Fix===
		+	For fixing protein gels
		+	*25% Isopropanol
		+	*65% Water
		+	*10% Acetic Acid
		+
		+	===Coomassie Rapid Stain===
		+	For staining protein gels
		+	*10% Acetic Acid
		+	*0.006% Coomassie G-250
		+	*90% Water
		+
		+	===Coomassie Brilliant Blue Solution===
		+	Reagent for [[Oneill_Lab:Protocols#Bradford Assay\|Bradford Assay]]
		+	#Dissolve 100mg Coomassie Brilliant Blue G-250 in 50ml 100% EtOH
		+	#Add 100mL 85% phosphoric acid
		+	#Bring volume to 1L with MilliQ
		+	#Filter through Whatman No. 1 paper
		+	#Store at 4 °C

	===20% SDS===		===20% SDS===

Seth Kasowitz — Mon, 30 Apr 2007 18:15:05 GMT

←Older revision		Revision as of 18:15, 30 April 2007
Line 56:		Line 56:
	*Add 32g NaOH / 2L		*Add 32g NaOH / 2L
	**NaOH cannot be stored in glass containers		**NaOH cannot be stored in glass containers
		+
		+	===X-Gal===
		+	*Dissolve X-Gal in dimethylformamide to final concentration of 40 mg/mL
		+	*Store at -20°C in a container shielded from light

	==Loading Dyes==		==Loading Dyes==
Line 173:		Line 177:
	#Adjust volume to 2L		#Adjust volume to 2L
	#Autoclave		#Autoclave
-
-	~~===Formaldehyde gel===~~
-	~~For making a 1% 100 mL~~
-	~~#Combine~~
-	~~#*5mL [[#20x Running Buffer\|20x Running Buffer]]~~
-	~~#*77mL deionized water~~
-	~~#*1g agarose~~
-	~~#Boil in microwave until agarose dissolves~~
-	~~#Add 18mL 37% formaldehyde~~
-	~~#*Swirl flask while pouring~~
-	~~#*Add formaldehyde in fume hood~~
-	~~#Pour gel in fume hood~~

Seth Kasowitz — Mon, 30 Apr 2007 16:39:41 GMT

Formaldehyde gel

←Older revision		Revision as of 16:39, 30 April 2007
Line 177:		Line 177:
	For making a 1% 100 mL		For making a 1% 100 mL
	#Combine		#Combine
-	#*5mL [[~~Oneill_lab:chemicals~~#20x Running Buffer\|20x Running Buffer]]	+	#*5mL [[#20x Running Buffer\|20x Running Buffer]]
	#*77mL deionized water		#*77mL deionized water
	#*1g agarose		#*1g agarose

Seth Kasowitz — Mon, 30 Apr 2007 16:39:10 GMT

RNA loading dye

←Older revision		Revision as of 16:39, 30 April 2007
Line 92:		Line 92:
	*450μL formamide		*450μL formamide
	*160μL 37% formaldehyde		*160μL 37% formaldehyde
-	*45μL [[~~Oneill_lab:chemicals~~#~~20x Running Buffer~~\|20x Running Buffer]]	+	*45μL [[#20x_Running_Buffer\|20x Running Buffer]]

	==PCR reagents==		==PCR reagents==

Seth Kasowitz — Mon, 30 Apr 2007 16:15:19 GMT

←Older revision		Revision as of 16:15, 30 April 2007
Line 92:		Line 92:
	*450μL formamide		*450μL formamide
	*160μL 37% formaldehyde		*160μL 37% formaldehyde
-	*45μL 20x Running Buffer	+	*45μL [[Oneill_lab:chemicals#20x Running Buffer\|20x Running Buffer]]

	==PCR reagents==		==PCR reagents==
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	*Vortex briefly		*Vortex briefly
	*Aliquot and store -20°C		*Aliquot and store -20°C
-	==Solutions ~~for Southern Blots~~==	+	==Blotting Solutions==
	===Depurination Solution===		===Depurination Solution===
	====Contents====		====Contents====
Line 163:		Line 163:

	''Note: SDS will precipitate at low temperatures. We have found keeping Church's Solution at 37°C may help in limiting instances of blot disease''		''Note: SDS will precipitate at low temperatures. We have found keeping Church's Solution at 37°C may help in limiting instances of blot disease''
		+
		+	===20x Running Buffer===
		+	For preparing gels for northern blotting
		+	#Dissolve in water
		+	#*167.4g MOPS
		+	#*27.2g Sodium Acetate
		+	#*80mL 0.5M EDTA pH 8.0
		+	#Adjust pH to 7.0 with 10N NaOH
		+	#Adjust volume to 2L
		+	#Autoclave
		+
		+	===Formaldehyde gel===
		+	For making a 1% 100 mL
		+	#Combine
		+	#*5mL [[Oneill_lab:chemicals#20x Running Buffer\|20x Running Buffer]]
		+	#*77mL deionized water
		+	#*1g agarose
		+	#Boil in microwave until agarose dissolves
		+	#Add 18mL 37% formaldehyde
		+	#*Swirl flask while pouring
		+	#*Add formaldehyde in fume hood
		+	#Pour gel in fume hood

Seth Kasowitz — Mon, 30 Apr 2007 15:49:07 GMT

Loading Dyes