Griffitts:Common buffers: Difference between revisions
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===colony lysis solution (for PCR) (100 mL)=== | ===colony lysis solution (for PCR) (100 mL)=== | ||
* 96.2 mL of sterile ddH<sub>2</sub>O | * 96.2 mL of sterile ddH<sub>2</sub>O | ||
* 0.5 mL of [[Griffitts:Common buffers#1 M Tris, pH 8.0 (75 mL)|1 M Tris | * 0.5 mL of [[Griffitts:Common buffers#1 M Tris, pH 8.0 (75 mL)|1 M Tris pH 8.0]] (final concentration: 5 mM) | ||
* 0.8 mL of [[Griffitts:Common buffers#0.25 M EDTA (75 mL)|.25 M EDTA]] (final concentration: 2 mM) | * 0.8 mL of [[Griffitts:Common buffers#0.25 M EDTA (75 mL)|.25 M EDTA]] (final concentration: 2 mM) | ||
* 2.5 mL of [[Griffitts:Common buffers#20% Triton X-100|20% Triton X-100]] (final concentration: 0.5%) | * 2.5 mL of [[Griffitts:Common buffers#20% Triton X-100|20% Triton X-100]] (final concentration: 0.5%) | ||
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Note: Store in fridge<br> | Note: Store in fridge<br> | ||
===TE buffer=== | ===TE buffer=== | ||
* 50 mM [[ | * 50 mM [[Griffitts:Common buffers#1 M Tris, pH 8.0 (75 mL)|1 M Tris pH 8.0]] | ||
* 10 mM [[ | * 10 mM [[Griffitts:Common buffers#0.25 M EDTA (75 mL)|.25 M EDTA]] | ||
===1 M Tris, pH 7.5 (75 mL)=== | ===1 M Tris, pH 7.5 (75 mL)=== | ||
* 75 mL ddH<sub>2</sub>O | * 75 mL ddH<sub>2</sub>O | ||
* 9.09 g Tris (Trizma) | * 9.09 g Tris (Trizma) | ||
* pH to 7.5 with HCl | * pH to 7.5 with HCl | ||
Autoclave | |||
===1 M Tris, pH 8.0 (75 mL)=== | ===1 M Tris, pH 8.0 (75 mL)=== | ||
* 75 mL ddH<sub>2</sub>O | * 75 mL ddH<sub>2</sub>O | ||
* 9.09 g Tris (Trizma) | * 9.09 g Tris (Trizma) | ||
* pH to 8.0 with HCl | * pH to 8.0 with HCl | ||
Autoclave | |||
===0.25 M EDTA (75 mL)=== | ===0.25 M EDTA (75 mL)=== | ||
* 75 mL ddH<sub>2</sub>O | * 75 mL ddH<sub>2</sub>O | ||
* 6.98 g EDTA | * 6.98 g EDTA | ||
* pH to 8.0 with NaOH | * pH to 8.0 with NaOH | ||
Autoclave | |||
===20% Triton X-100=== | ===20% Triton X-100=== | ||
Note: do not filter sterilize<br> | Note: do not filter sterilize<br> |
Revision as of 10:51, 13 February 2008
Electrophoresis Buffers
2% bromophenol blue
Sterile filter
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
DNA ladder
- 147 μL ddH20
- 34 μL 8X DNA loading buffer
- 20 μL 1 kb ladder from NEB
- 2 μL 0.25M EDTA
Note: use 5 μL per lane; 3kb band=60 ng
Protein ladder
- Open vial of Sigma SDS6H2-1VL
- Add 1 mL ddH20
- Add 500 μL 6X SDS loading
- Aliquot 50 μL per tube and store at -20
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
Coomassie destain solution (1 L)
- 550 mL ddH20
- 400 mL ethanol
- 50 mL acetic acid
DNA extraction buffers
colony lysis solution (for PCR) (100 mL)
- 96.2 mL of sterile ddH2O
- 0.5 mL of 1 M Tris pH 8.0 (final concentration: 5 mM)
- 0.8 mL of .25 M EDTA (final concentration: 2 mM)
- 2.5 mL of 20% Triton X-100 (final concentration: 0.5%)
- in fridge with the antibiotics
- in fridge with the antibiotics
Note: Store in fridge
TE buffer
- 50 mM 1 M Tris pH 8.0
- 10 mM .25 M EDTA
1 M Tris, pH 7.5 (75 mL)
- 75 mL ddH2O
- 9.09 g Tris (Trizma)
- pH to 7.5 with HCl
Autoclave
1 M Tris, pH 8.0 (75 mL)
- 75 mL ddH2O
- 9.09 g Tris (Trizma)
- pH to 8.0 with HCl
Autoclave
0.25 M EDTA (75 mL)
- 75 mL ddH2O
- 6.98 g EDTA
- pH to 8.0 with NaOH
Autoclave
20% Triton X-100
Note: do not filter sterilize
Store in fridge with the antibiotics
plasmid purification solutions
resuspension buffer
- 50 mM Tris pH 8.0
- 10 mM EDTA
lysis solution
- 1% SDS
- 200 mM NaOH
neutralization solution
- 4 M KOAc (pH to 5.5 with acetic acid)
RNAse A
- 5 mg/mL RNAse A (in freezer)
- 10 mM Tris pH 8.0
Note: RNAse A is purchased from Sigma (R-6513)
silica suspension
- Wash 5X with ddH20
- Resuspend as 50% slurry in ddH20
Store at -20°C
silica wash solution
- 50% ethanol
- 10 mM Tris pH 7.5
- 50 mM NaCl
- 2.5 mM EDTA
Proteinase K
- 20 mg/mL Proteinase K (in freezer)
- 20 mM Tris pH 8.0
Store at -20°C
Lysozyme
- 1 mL dH2O
- 30mg lysozyme (in freezer)
- Dilute 60X into lysis buffer
Store at -20°C