Oneill Lab:Chemicals

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	*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==

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Revision as of 12:39, 30 April 2007

Michael J. O'Neill Lab University of Connecticut Department of Molecular and Cell Biology

Contents 1 Common Lab Chemical Recipes 1.1 Phosphate Buffered Saline 1.2 20x SSC 1.3 20x TBE-modified 1.4 50x TAE 1.5 20% SDS 1.6 0.5M EDTA 1.7 1M Tris 1.8 1M HCl 1.9 1M NaOH 1.10 0.4M NaOH 2 Loading Dyes 2.1 6x OrangeG 2.2 10x BlueJuice 2.3 5x RedJuice 2.3.1 Contents 2.3.2 Preparation 2.4 RNA loading dye 3 PCR reagents 3.1 10x PCR Buffer 3.1.1 Contents 3.1.2 Preparation 3.2 10mM dNTP Mix 3.2.1 Preparation 4 Blotting Solutions 4.1 Depurination Solution 4.1.1 Contents 4.1.2 Preparation 4.2 Denaturation Solution 4.2.1 Contents 4.2.2 Preparation 4.3 Neutralization Solution 4.3.1 Contents 4.3.2 Preparation 4.4 Church's Solution 4.4.1 Materials 4.4.2 Procedure 4.5 20x Running Buffer 4.6 Formaldehyde gel

Common Lab Chemical Recipes

The recipes listed here can be found in the grey protocol binder

Phosphate Buffered Saline

1. Add 1 tablet per 100 ml diH₂O
2. Autoclave

20x SSC

Recipe to make 4L

• Dissolve 701.28g NaCl and 352.92g NaCitrate
• pH to 7.0
• Bring final volume to 4L
• Autoclave if necessary

20x TBE-modified

Recipe makes 2L

• 649.096g Tris
• 185.24g Boric Acid
• 37.968g EDTA
• Bring final volume to 2L

50x TAE

• 242g Tris
• 37.2g EDTA
• 57.1mL Glacial Acetic Acid
• Bring final volume to 1L with diH₂O

20% SDS

• 200g SDS / 1L
• Wear mask and safety goggles!
• Heat to 68°C to dissolve

0.5M EDTA

• Dissolve 186.1g EDTA / 1L
• pH to 8.0
  • May take several mL of 10N NaOH to adjust pH
  • EDTA will not all go into solution until pH is adjusted
• Autoclave if necessary

1M Tris

• Dissolve 60.55g Tris / 500 mL
• Adjust pH as desired (7.5-8.0 typically)

1M HCl

• Add 8.3mL 12M HCl / 100 mL
  • Always add acid to water

1M NaOH

• Add 2g NaOH / 50 mL
  • NaOH cannot be stored in glass containers

0.4M NaOH

• Add 32g NaOH / 2L
  • NaOH cannot be stored in glass containers

Loading Dyes

6x OrangeG

• 50mg OrangeG
• 1.5g Ficoll
• Add MilliQ water to 10 mL
• Aliquot and store at 4°C

10x BlueJuice

• 3.25g Sucrose (65% Final)
• 50μL 1M Tris (10mM Final)
• 100μL 0.5M EDTA (10mM Final)
• 15mg bromophenol blue
• 15mg Xylene cyanol
• Bring volume to 5 mL with MilliQ water
• Aliquot and store at room temperature

5x RedJuice

RedJuice can be safely added to PCR reactions

Contents

• 60% Sucrose
• 1mM Cresol Red (FW= 404.4)

Preparation

• 12g sucrose
• Add MilliQ to 20mL and mix
• Pass through 0.45μm filter to sterilize
• Add 8.09 mg cresol red
• Aliquot and store at 4°C

RNA loading dye

• 50μL glycerol
• 25μL 1% bromophenol blue
• 25μL 1% xylene cyanol
• 5μL 0.5M EDTA
• 450μL formamide
• 160μL 37% formaldehyde
• 45μL 20x Running Buffer

PCR reagents

10x PCR Buffer

Contents

100mM Tris pH 8.4 500mM KCl 15mM MgCl₂

Preparation

Makes 25mL

• 2.5mL 1M Tris
• 375μL 1M MgCl₂
• 12.5mL 1M KCl
• 9.625mL diH₂O

Aliquot and store -20°C

10mM dNTP Mix

Preparation

• 10μL dATP
• 10μL dCTP
• 10μL dTTP
• 10μL dGTP
• 60μL MilliQ H₂O
• Vortex briefly
• Aliquot and store -20°C

Blotting Solutions

Depurination Solution

Contents

1L 0.25M HCl

Preparation

• Dilute 22mL 12M HCl in 978 mL diH₂O
• Label and store at room temperature

Denaturation Solution

Contents

0.5M NaOH 1.5M NaCl

Preparation

• 87.66g NaCl
• 20.00g NaOH
• Bring final volume to 1L
  • Store in a labelled plastic bottle
  • Should be made fresh

Neutralization Solution

Contents

1.5M NaCl 0.5M Tris 2mM EDTA pH 8.0

Preparation

• 87.66g NaCl
• 60.56g Tris
• 4mL EDTA pH 8.0
• 800 mL diH₂O
• Adjust pH to 7.2
  • Will take about 34 mL 12M HCl
• Bring volume to 1L with diH₂O

Church's Solution

Materials

• 20% SDS
• 0.5M EDTA pH=8.0
• 1M Na₂HPO₄
• diH₂O

Procedure

1. Mix 35ml SDS and 0.2ml EDTA
2. Add 50ml Na₂HPO₄
3. Bring final volume to 100ml

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

1. Dissolve in water
   • 167.4g MOPS
   • 27.2g Sodium Acetate
   • 80mL 0.5M EDTA pH 8.0
2. Adjust pH to 7.0 with 10N NaOH
3. Adjust volume to 2L
4. Autoclave

Formaldehyde gel

For making a 1% 100 mL

1. Combine
   • 5mL 20x Running Buffer
   • 77mL deionized water
   • 1g agarose
2. Boil in microwave until agarose dissolves
3. Add 18mL 37% formaldehyde
   • Swirl flask while pouring
   • Add formaldehyde in fume hood
4. Pour gel in fume hood