Griffitts:Common buffers

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Contents

Electrophoresis Buffers

2% bromophenol blue

Sterile filter

8X DNA loading

6X SDS loading

6X NAT loading

10X NAT running

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

DNA ladder

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 T3E0.3:

Note: T3E0.3 is what we use most of the time in the lab
For 75 mL T5E0.5:

For 75 mL T10E1:

For 75 mL T50E10:

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

lysis solution

  • 1% SDS
  • 200 mM NaOH

Neutralization solution

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

RNAse A

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

Proteinase K

Store at -20°C

Lysozyme

  • 1 mL dH2O
  • 30mg lysozyme (in freezer)
  • Dilute 60X into lysis buffer

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

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