How to prepare 1M Tris, 1%Agarose, 10% SDS, 0.5M EDTA, 5X Tris- borate, 50X Tris-acetate TAE, Tracking Dye for Electrophoresis

1M Tris

  • 121.1 g.   Tris base
  • 800 ml.    ddH2O
  • Adjust the pH to the desired value by adding concentrated HCl.

pH                HCl

7.4                70 ml

7.6                60 ml

8.0                42 ml

  • Allow the solution to cool to room temperature before making final adjustments to the pH.  Adjust the volume of the solution to 1 liter with H2O. Aliquot into containers and sterilize by autoclaving.

Comments

If the 1 M solution has a yellow color, discard it and obtain a better quality Tris.

Although many types of electrodes do not accurately measure the pH of Tris solutions, suitable electrodes can be obtained from most manufacturers.

The pH of Tris solutions is temperature-dependent and decreases approximately 0.03 pH units for each 1oC increase in temperature.  For example, a 0.05 M solution has pH values of 9.5, 8.9, and 8.6 at 5 oC, 25 oC, and 37 oC respectively.

1%Agarose

  • 1 g.          agarose
  • 100ml.     0.5X TBE Buffer

10% SDS

  • (Sodium Dodecyl Sulfate also known as sodium lauryl sulfate)
  • 100 g.     SDS (eletrophoresis-grade)
  • 900 ml    ddH2O
  • Mix and heat to 68OC to dissolve. Adjust the pH to 7.02 by adding a few drops of concentrated HCl.  Adjust the volume to l liter with ddH2O.  Aliquot into containers.

Comments

Wear a mask when weighing SDS and wipe down the weighing area and balance after use, because the fine crystals of SDS disperse easily.  Does not need autoclaving.

0.5M EDTA (pH 8.0)

  • 186.1 g.    Disodium ethylenediaminetetraacetate·2H2O
  • 800ml       ddH2O
  • Stir vigorously on a magnetic stirrer.  Adjust the pH to 8.0 with NaOH (~20 g of NaOH pellets).  Aliquot and sterilize by autoclaving.

Comments

The disodium salt of EDTA will not go into solution until the pH of the solution is adjusted to approximately 8.0 by the addition of NaOH.

Lysis Buffer

Final Concentration

  • 20 ml      10% SDS                                                     (1%)
  • 2 ml         IM Tris · HCl (pH 8.0)                                (10mM)
  • 4 ml         0.5M EDTA (pH8.0)                                  (10mM)
  • 5.84 g      NaCl                                                           (0.5M)
  • Bring up to 200ml with ddH2O. Dispense into bottles.

5X Tris- borate (TBE)

  • 54 g          Tris Base (or Sigma 7-9)
  • 27.5 g.      Boric acid
  • 20 ml.       0.5M EDTA (pH 8.0)
  • 800 ml      ddH2O
  • Stir to get into solution.  Bring up to 1 liter with ddH2O.  Does not need to be autoclaved.  Remove stir bar.

TE Buffer (pH 8.0)

Final Concentration

  • 2 ml        1M Tris HCl (pH 8.0)                                  (10 mM)
  • 0.4 ml     0.5M EDTA (pH 8.0)                                  (1 mM)
  • 200 ml    ddH2O

50X Tris-acetate (TAE)

  • 242 g.        Tris Base
  • 57.1 ml      Glacial acetic acid
  • 100 ml       0.5M EDTA (pH 8.0)
  • 1000 ml ddH2O

Tracking Dye for Electrophoresis

  • O.25% bromophenol blue
  • 40% (w/v) sucrose in water

Commonly used Gel Loading Buffers

Loading buffers are solutions of high density that enable samples to be introduced easily into gel slots.  They also contain one or more tracking dyes that allow the progress of the electrophoresis to be monitored easily.

Type I 6x Buffer

0.25% bromophenol blue

0.25% xylene cyanol

40% (w/v) sucrose in ddH2O

Store at 4oC

Type II 10x Buffer                                                           For 10ml

0.25% bromophenol blue                                25mg bromophenol blue

0.25% xylene cyanol

25% Ficoll (type 400) in ddH2O                     2.5g Ficoll

Store at room temperature

Type III 6x Buffer

0.25% bromophenol blue

0.25% xylene cyanol

30% glycerol in ddH2O

Store at 4oC

Type IV 6x Buffer

0.25% bromophenol blue

40% (w/v) sucrose in ddH2O

Store at 4oC

Glycerol buffer for Pryor Lab (TypeIII)

30% Glycerol in ddH20

200 ul of 2.5% dye stock solution

Add 3mls glycerol to a 15ml sterile centrifuge tube.  Add 200ul dye stock solution.  Bring up to 10 ml with ddH20.

2.5% Dye Stock Solution

250 mg    Bromphenol blue

10 ml        ddH20

Mixed in a screw cap glass test tube. Cover with foil.  Stored in the VWR refrigerator.

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