Griffitts:Common buffers: Difference between revisions
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* 400 mL [[Wikipedia:ethanol|ethanol]] | * 400 mL [[Wikipedia:ethanol|ethanol]] | ||
* 50 mL [[Wikipedia:acetic acid|acetic acid]] | * 50 mL [[Wikipedia:acetic acid|acetic acid]] | ||
==DNA extraction buffers== | |||
===colony lysis solution (for PCR)=== | |||
* 5 m[[Wikipedia:Concentration#Molarity|M]] [[Wikipedia:Tris|Tris]] pH 8.0 | |||
* 2 m[[Wikipedia:Concentration#Molarity|M]] [[Wikipedia:EDTA|EDTA]] | |||
* 0.5% [[Wikipedia:Triton X-100|Triton X-100]] | |||
==plasmid purification solutions== | |||
===resuspension buffer=== | |||
* 50 m[[Wikipedia:Concentration#Molarity|M]] [[Wikipedia:Tris|Tris]] pH 8.0 | |||
* 10 m[[Wikipedia:Concentration#Molarity|M]] [[Wikipedia:EDTA|EDTA]] | |||
===lysis solution=== | |||
* 1% [[Wikipedia:Sodium dodecyl sulfate|SDS]] | |||
* 200 m[[Wikipedia:Concentration#Molarity|M]] NaOH | |||
===neutralization solution=== | |||
* 4 [[Wikipedia:Concentration#Molarity|M]] KOAc pH 5.5 (acetic acid) | |||
===[[Wikipedia:Ribonuclease A|RNAse A]]=== | |||
* 5 mg/mL [[Wikipedia:Ribonuclease A|RNAse A]] | |||
* 50 m[[Wikipedia:Concentration#Molarity|M]] [[Wikipedia:Tris|Tris]] pH 8.0 | |||
===silica suspension=== | |||
Wash 5X with ddH<sub>2</sub>0<br> | |||
Resuspend as 50% slurry in ddH<sub>2</sub>0 | |||
===silica wash solution=== | |||
* 50% [[Wikipedia:ethanol|ethanol]] | |||
* 10 m[[Wikipedia:Concentration#Molarity|M]] [[Wikipedia:Tris|Tris]] pH 7.5 | |||
* 50 m[[Wikipedia:Concentration#Molarity|M]] NaCl | |||
* 2.5 m[[Wikipedia:Concentration#Molarity|M]] [[Wikipedia:EDTA|EDTA]] |
Revision as of 16:16, 7 September 2007
Electrophoresis Buffers
8X DNA loading
- 40% glycerol
- 1/50 dilution of bromophenol blue
6X SDS loading
- 7.5 mL 80% glycerol
- 2.5 mL 1M Tris pH 6.8
- 1.2 g SDS
- 600 μL 2-mercaptoethanol
- 150 μL 2% bromophenol blue
2% bromophenol blue
Sterile filter
DNA ladder
- 147 μL ddH20
- 34 μL 8X DNA loading buffer
- 20 μL 1 kb ladder from NEB
- 2 μL 0.25 M EDTA
Note: use 5 μL per lane
Protein ladder
- 1 mL ddH20
- 500 μL 6X SDS loading
- 1 μL ladder from Sigma
Note: use 5 μL per lane
ethidium bromide (10 mL)
- 10 mL ddH20
- 10 mg ethidium bromide
Note: use 8 μL per 50 mL gel
50X TAE (1 L)
- ~800 mL ddH20
- 242 g Trizma base
- 57.1 mL glacial acetic acid
- 100 mL 0.25 M EDTA (pH 8.0)
QS to volume with ddH20
Note: this is half the amount of EDTA compared to standard TAE
10X Laemmli running buffer
QS to volume with ddH20
Coomassie stain (300 mL)
- 120 mL ethanol
- 0.3 g Brilliant Blue R
Mix for 30 min.
- 30 mL acetic acid
- 150 mL ddH20
destain solution (1 L)
- 550 mL ddH20
- 400 mL ethanol
- 50 mL acetic acid
DNA extraction buffers
colony lysis solution (for PCR)
- 5 mM Tris pH 8.0
- 2 mM EDTA
- 0.5% Triton X-100
plasmid purification solutions
resuspension buffer
lysis solution
neutralization solution
- 4 M KOAc pH 5.5 (acetic acid)
RNAse A
silica suspension
Wash 5X with ddH20
Resuspend as 50% slurry in ddH20