27 Feb 2012

Separation of DNA Fragments Using PAGE Method

This method has the capacity to separate DNA fragments using the size of as small as 10 bp or longer to 1 kb with all the resolution regarding as little as 1 bp. While agarose gel electrophoresis is just capable of distinct DNA fragments while using bigger dimension of which PAGE really does or even in the size selection of 100 nucleotides to help close to 10 - 15 kb.


Gel apparatus: Quite a few designs connected with equipment are generally retail offered. This gel is actually poured involving two vertical plates presented apart by spacers

The plates ought to be cleansed extensively then wiped having ethanol. To aid make sure your gel simply sticks to one plate if your device is disassembled, implement silicon to be able to one of several gel plates. This is easily completed by means of wiping the actual plate which has a cells soaked with dimethyl dichlorosilane solution and cleaning the actual plate within distilled water accompanied by ethanol. If the plates usually are baked with 100oC intended for 30 minutes, this siliconization lasts 4 to five gel runs.

Deionized WATER: Autoclaved h2o isn't essential for the gel mixture or perhaps running buffer, nevertheless it should be used by diluting samples as well as purification coming from gel slices.
10x TBE: 108 g associated with Trizma base (Tris), 55 g associated with boric acid, as well as 9. 3 g involving ethylenediaminetetraacetic acid (EDTA) (disodium sodium ). Constitute to help I L solution along with deionized WATER, which should become discarded each time a precipitate types.
Acrylamide stock: 30% acrylamide, 1% N, N'-methylene bisacrylamide. Store from 4oC. This can be obtainable over the counter, or it can be made by dissolving acrylamide along with bisacrylamide throughout water, which should become filtered. Acrylamide is really a neurotoxin and as a consequence need to be managed carefully. Hand protection and a mask has to be worn while weighing out.
APS: 10% Ammonium persulphate (w/v). This is stashed with 4oC with regard to 1-2 a few months.
TEMED: N, N, N', N'-tetramethyl-l, 2-diaminoethane. Keep on 4oC.
5X sample buffer: 15% Ficoll solution, 2. 5X TBE, 0. 25% (w/v) xylene cyanol along with 0. 025% (w/v) bromophenol blue.
Ethidium bromide: A l0-mg/mL solution. Ethidium bromide is a potent mutagen and will always be managed after due thought. Shop with 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.

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.

Satisfied separating DNA fragments.

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