Thursday, September 24, 2009

Separation of DNA Fragments Using PAGE Method

This method is able to separate DNA fragments with the size of as small as 10 bp and up to 1 kb with the resolution of as little as 1 bp. While agarose gel electrophoresis is only able to separate DNA fragments with the bigger size that PAGE does or in the size range of 100 nucleotides to around 10 – 15 kb.

  • Gel apparatus: Many designs of apparatus are commercially available. The gel is poured between two vertical plates held apart by spacers

    The plates should be cleaned thoroughly and then wiped with ethanol. To help ensure that the gel only sticks to one plate when the apparatus is disassembled, apply silicon to one of the gel plates. This is easily done by wiping the plate with a tissue soaked in dimethyl dichlorosilane solution and then washing the plate in distilled water followed by ethanol. If the plates are baked at 100oC for 30 min, the siliconization will last four to five gel runs.

  • Deionized H2O: Autoclaved water is not necessary for the gel mix or running buffer, but it should be used for diluting samples and purification from gel slices.
  • l0x TBE: 108 g of Trizma base (Tris), 55 g of boric acid, and 9.3 g of ethylenediaminetetraacetic acid (EDTA) (disodium salt). Make up to I L solution with deionized H2O, which should be discarded when a precipitate forms.
  • Acrylamide stock: 30% acrylamide, 1% N,N'-methylene bisacrylamide. Store at 4oC. This is available commercially, or it can be made by dissolving acrylamide and bisacrylamide in water, which should be filtered. Acrylamide is a neurotoxin and therefore must be handled carefully. Gloves and a mask must be worn when weighing out.
  • APS: 10% Ammonium persulphate (w/v). This can be stored at 4oC for 1-2 months.
  • TEMED: N,N,N',N'-tetramethyl-l,2-diaminoethane. Store at 4oC.
  • 5X sample buffer: 15% Ficoll solution, 2.5X TBE, 0.25% (w/v) xylene cyanol and 0.025% (w/v) bromophenol blue.
  • Ethidium bromide: A l0-mg/mL solution. Ethidium bromide is a potent mutagen and should be handled with care. Store at 4oC in the dark.
  • For 50 mL, enough for a 18 x 14 x 0.15 cm gel, mix l0x TBE, acrylamide, H2O, and APS as described in Table below.
  • Table preparation of Polyacrylamide Gel Mixes
  • Just prior to pouring, add 50 microliters of TEMED and mix by swirling.
  • Immediately pour the gel mix between the gel plates and insert the gel comb. Leave to set; this takes about 30 min.
  • Fill the gel apparatus with 0.5X TBE and remove the comb. Use a syringe to wash out the wells, this may take multiple washes. It is important to remove as much unpolymerized acrylamide as possible because this impairs the running in of the samples

    If you are separating very small fragments, e.g., less than 50 bp, the gel should be prerun for 30 min, as this elevates the resolution problem experienced with fragments running close to the electrophoresis front..

  • Add 0.2 volume of 5x sample buffer to each sample, usually in 10-20 microliters of TE, water, or enzyme buffer. Mix and spin the contents to the bottom of the tube

    High-salt buffers (above 50 mM NaCl) will affect sample mobility and tend to make bands collapse. In this case, salt should be removed by ethanol precipitation..

  • Load the samples on the gel and run at 200-300 V (approximately 10 V/cm) until the bromophenol blue band is two-thirds of the way down the gel; this takes about 2.5 h

    Do not run the gel faster than 10 V/cm, as this will cause the gel to overheat, affecting the resolution. The gel can be run more slowly, e.g., 75 V will run overnight.

  • Disassemble the gel apparatus and place the gel to stain in I mg/mL of ethidium bromide for approximately 30 min. View the stained gel on a transilluminator.
Happy separating DNA fragments.

Reference: Number 20 on References