Oneill Lab:Chemicals

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Current revision (10:04, 17 October 2007) (view source)
(Blotting Solutions)
 
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*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
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*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
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*15.1g Tris
 +
*94g Glycine
 +
*25ml 20% SDS
 +
*Adjust volume to 500 mL with water
 +
*Store at 4&deg;C
 +
 +
===Isopropanol Fix===
 +
For fixing protein gels
 +
*25% Isopropanol
 +
*65% Water
 +
*10% Acetic Acid
 +
 +
===Coomassie Rapid Stain===
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For staining protein gels
 +
*10% Acetic Acid
 +
*0.006% Coomassie G-250
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*90% Water
 +
 +
===Coomassie Brilliant Blue Solution===
 +
Reagent for [[Oneill_Lab:Protocols#Bradford Assay|Bradford Assay]]
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#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 &deg;C
===20% SDS===
===20% SDS===
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*Add 32g NaOH / 2L
*Add 32g NaOH / 2L
**NaOH cannot be stored in glass containers
**NaOH cannot be stored in glass containers
 +
 +
===ReagentB===
 +
*TEN
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**0.1M NaCl
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**10mM Tris pH 8.0
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**1mM EDTA pH 8.0
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*1% SDS
 +
Store at room temperature
 +
 +
===X-Gal===
 +
*Dissolve X-Gal in dimethylformamide to final concentration of 40 mg/mL
 +
*Store at -20&deg;C in a container shielded from light
==Loading Dyes==
==Loading Dyes==
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*450&mu;L formamide
*450&mu;L formamide
*160&mu;L 37% formaldehyde
*160&mu;L 37% formaldehyde
-
*45&mu;L [[Oneill_lab:chemicals#20x Running Buffer|20x Running Buffer]]
+
*45&mu;L [[#20x_Running_Buffer|20x Running Buffer]]
==PCR reagents==
==PCR reagents==
Line 163: Line 211:
''Note: SDS will precipitate at low temperatures. We have found keeping Church's Solution at 37&deg;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&deg;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 &mu;L 2M MgCl<sub>2</sub>
 +
*100 &mu;L 5M NaCl
 +
*50 &mu;L 1M DTT
 +
*2.5 mL 2M HEPES
 +
*Bring volume to 5mL with diH<sub>2</sub>O
===20x Running Buffer===
===20x Running Buffer===
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#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==
 +
[[User:Seth|Seth Kasowitz]] [mailto:seth.kasowitz@uconn.edu]
-
===Formaldehyde gel===
+
[http://www.oneill.mcb.uconn.edu/Michael/MONEILLhome.html|O'Neill Lab Homepage]
-
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
+

Current revision

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

Contents

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

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 NaN3
    • 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 Bradford Assay

  1. Dissolve 100mg Coomassie Brilliant Blue G-250 in 50ml 100% EtOH
  2. Add 100mL 85% phosphoric acid
  3. Bring volume to 1L with MilliQ
  4. Filter through Whatman No. 1 paper
  5. Store at 4 °C

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

ReagentB

  • TEN
    • 0.1M NaCl
    • 10mM Tris pH 8.0
    • 1mM EDTA pH 8.0
  • 1% SDS

Store at room temperature

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

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

5x Random Priming Buffer

Components

  • 250 mM Tris pH 8.0
  • 25 mM MgCl2
  • 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 MgCl2
  • 100 μL 5M NaCl
  • 50 μL 1M DTT
  • 2.5 mL 2M HEPES
  • Bring volume to 5mL with diH2O

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. Filter sterilize

This buffer is light sensitive. Store at room temp in a foil wrapped or otherwise light shielded container.

Links and Contacts

Seth Kasowitz [1]

Lab Homepage

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