Griffitts:Common buffers

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Electrophoresis Buffers

2% bromophenol blue

• 0.4 g BPB
• 20 mL 50 mM Tris pH 8.0
  

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

• 30.3 g Tris base
• 144.2 g glycine
• 10 g 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) (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
    

Note: Store in fridge

TE buffer

• 50 mM Tris pH 8.0
• 10 mM 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)
• 50 mM or 20 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