Griffitts:Common buffers

Electrophoresis Buffers

2% bromophenol blue

• 0.4 g bromophenol blue (BPB)
• 20 mL 50 mM Tris pH 8.0
  

Sterile filter

8X DNA loading

• 40% glycerol
• 1/50 dilution of 2% bromophenol blue

6X SDS loading

• 7.5 mL 80% glycerol
• 2.5 mL 1 M Tris pH 6.8
• 1.2 g SDS
• 600 μL 2-mercaptoethanol (in fume hood)
• 150 μL 2% bromophenol blue

6X NAT loading

• 7.5 mL 80% glycerol
• 2.5 mL 1 M Tris pH 6.8
• 5 μL 2-mercaptoethanol (in fume hood)
• 150 μL 2% bromophenol blue

10X NAT running

• 3 g Tris Base
• 14.4 g glycine
• dH₂O to 100 mL

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; 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°C
  

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 (500 mL)

• 400 mL ddH₂0
• 121 g Trizma base
• 28 mL glacial acetic acid
• 4 g Na₂EDTA
  

Stir until EDTA is dissolved.
Final pH should be between 8.1 and 8.4
Note: this is half the amount of EDTA compared to standard TAE.

10X Laemmli running buffer (1 L)

• 30.3 g Trizma base (Tris)
• 144.2 g glycine
• 10 g sodium dodecyl sulfate (SDS)

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) (75 mL)

• 72.15 mL of sterile ddH₂O (i.e. remove 2.85 mL from a 75-mL bottle of sterile ddH₂O)
• 375 μL of 1 M Tris pH 8.0 (final concentration: 5 mM)
• 600 μL of .25 M EDTA (final concentration: 2 mM)
• 1.875 mL of 20% Triton X-100 (final concentration: 0.5%)
  • in fridge with the antibiotics
    

Note: Store in fridge

TE buffer

For 75 mL T₃E_0.3:

• 74.685 mL ddH₂O (i.e. remove 315 μL from 75 mL ddH₂O)
• 225 μL 1 M Tris pH 8.0
• 90 μL .25 M EDTA

Note: T₃E_0.3 is what we use most of the time in the lab
For 75 mL T₅E_0.5:

• 74.475 mL ddH₂O (i.e. remove 525 μL from 75 mL ddH₂O)
• 375 μL 1 M Tris pH 8.0
• 150 μL .25 M EDTA

For 75 mL T₁₀E₁:

• 73.95 mL ddH₂O (i.e. remove 1.05 mL from 75 mL ddH₂O)
• 750 μL 1 M Tris pH 8.0
• 300 μL .25 M EDTA

For 75 mL T₅₀E₁₀:

• 68.25 mL ddH₂O (i.e. remove 6.75 mL from 75 mL ddH₂O)
• 3.75 mL 1 M Tris pH 8.0
• 3 mL .25 M EDTA

1 M Tris, pH 6.8 (75 mL)

• 75 mL ddH₂O
• 9.09 g Tris (Trizma base)
• pH to 6.8 with HCl

Autoclave

1 M Tris, pH 7.5 (75 mL)

• 75 mL ddH₂O
• 9.09 g Tris (Trizma base)
• 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

1 M Tris, pH 8.8 (75 mL)

• 75 mL ddH₂O
• 9.09 g Tris (Trizma)
• pH to 8.8 with HCl

Autoclave

0.25 M EDTA (75 mL)

• 75 mL ddH₂O
• 6.98 g EDTA
• pH to 8.0 with NaOH

Autoclave

10% Triton X-100 (40 mL)

• 36 mL dH₂O
• 4 mL Triton X-100

Note: Stock solution of Triton X-100 is very viscous, so dispense slowly
Note: do not filter sterilize
Store at 4°C

20% Triton X-100 (40 mL)

• 32 mL dH₂O
• 8 mL Triton X-100

Note: Stock solution of Triton X-100 is very viscous, so dispense slowly
Note: do not filter sterilize
Store at 4°C

0.5 M MOPS (40 mL)

• 20 mL ddH₂O
• 4.19 g MOPS
• pH to 7.0 (~3.5 mL NaOH)

QS to volume with ddH₂O

plasmid purification solutions

Resuspension Buffer

• 68.25 mL ddH₂O (i.e. remove 6.75 mL from 75 mL ddH₂O)
• 3.75 mL 1 M Tris pH 8.0
• 3 mL .25 M EDTA

lysis solution

• 1% SDS
• 200 mM NaOH

Neutralization solution

• 4 M potassium acetate
• pH to 5.5 with acetic acid

RNAse A

• 20 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
• 100mg lysozyme (in freezer)

Store at -20°C

Bases

2N KOH (40 mL)

• 4.49 g KOH pellets
• 40 mL ddH₂O

NOTE: for KOH 2N = 2M

2N NaOH (40 mL)

• 3.2 g NaOH pellets
• 40 mL ddH₂O

NOTE: for NaOH 2N = 2M