Agarose Gel Electrophoresis of DNA

1) Dissolve 1 g of agarose in 100 ml of 1X TAE or TBE buffer (gives a 1% gel). See note for making LMP agarose gel.

2) Cast the gel with the comb in place.

3) Add 6X gel loading buffer to sample and load the samples into wells.

4) Run the gel submerged in 1X TAE or TBE (30-60 min. at 100-150V)

5) Stain the gel with ethidium bromide solution (10 μl per 100ml of buffer) for 10 - 15 min.

6) View on a long wave UV transilluminator.

note: a) TAE is better for cloning work as the borate in TBE may affect subsequent ligation reaction if it is not removed well. See FMC bioproducts for more details on comparison of buffers. TTE (Tris Taurine EDTA) may be used as an alternative.b) The percentage of gel used depend on the size of DNA to be separated. Low percentage gel separates high m.w. DNA while high percentage gel separates low m.w DNA.c) Gel may be cast with ethidium bromide solution added. This will save time staining but the gel will also run slightly slower (note that ethidium bromide is a powerful mutagen, so be careful in its use).d) In general the supercoiled circular plasmid run faster than cut plasmid. Multiple bands can be seen with closed circular plasmid (supercoiled DNA, relaxed DNA, denatured DNA, catenae, etc.).e) The distance tranvelled in a gel by a DNA molecule has a roughly logarithmic relationship with the m.w. of the DNA. f) Higher concentration of DNA may retard the movement of the DNA band. Higher concentration of salts in the buffer may also have the same effect.g) The RNA normally runs well in front of the DNA. Chromosomal DNA may be stuck in the well or move very slowly if present.h) Smear in the lane may indicates the degradation of the DNA by nucleases or maybe RNA. See also trouble-shootingi) To prepare low m.p agarose gel - first pour 50 ml (volume depends on gel apparatus used) of 2% of usual agarose (not LMP), let it set, put comb in (leave a small gap between gel surface and comb) and pour 100ml of ~1% LMP agarose. This method helps the low m.p agarose to set and provide a firm base for the handling of the gel.j) When loading DNA sample into the well of your gel, if the DNA sample is contaminated with ethanol or mineral oil, or if the gel is not completely submerged with buffer, the DNA solution may move out of the well. If this is a problem, use more loading buffer, or prepare the loading buffer in TAE instead of water, or use glycerol in the buffer and mix well, or make sure that your DNA sample is free of ethanol.k) Note that UV can damage your DNA. Although the UV transilluminator should be operating at a wavelength safer for your DNA, it is generally a good idea not to expose your DNA to UV light for too long. Overexposure to UV can result in ligation failure. It has been suggested that addition of 1-10mM cytodine or guanosine in gel can protect against UV damage.l) Electrophoresis of the gel can be done at constant current or constant voltage, however, it is safer to use constant voltage.m) You can destain the gel after staining (10-15 min. in water), but it is generally unncessary but would be useful if the band is faint.n) Methods of extracting DNA from gel: there are many methods for extracting DNA from gel apart from using commericial kits, some methods however yield cleaner DNA than others. In general, use LMP agarose if you wish to use the DNA for further manipulation.

Freeze squeeze - Excise band and place pieces of it in a home-made spin-column (a piece of filter placed in a PCR 0.5ml eppendorf, pierced the bottom of the PCR tube with a needle). Place The PCR tube at -20 °C for 15-20 mins or freeze in liquid N2. Then place the PCR tube into the 1.5 ml eppendorf and spin at top speed in a microfuge for 15 mins, collect DNA solution which can be or use directly. Syringe squeeze - Li and Ownby, (1993) BioTechniques 15,976-978 Powdered silica - see bionet.molbio.methds-reagnts FAQ (also useful for lots of other information on molecular biology. Melt excised bands at 70°C cool and add equal volume of TE-buffered phenol and mix. Spin for 5 min, remove aqueous phase, repeat and EtOh ppt. the DNA. See also phenol-base method . Electroelution - cut a small trough just ahead of the DNA band, place DEAE-cellulose paper, or dialysis tubing, or affinity membrane, or just buffer containing 0.3 M NaOAc and 10% sucrose into the trough and run the gel at 150 volt for a minute or two until all the DNA has gone into the trough. Collect DNA (then spun out in a home-made spin-column if it's trapped in paper) and ethanol precipitate. Kits - gives clean DNA for further manipulation. e.g. Geneclean (Bio 101), Prep-a-gene (Bio-rad), Qiaex (Qiagen), GELase (Epicentre), etc. Some work better than others.

Buffers: 50x TAE (per litre)242 g Tris base, 57.1 g glacial acetic acid, 100 ml 0.5 M EDTA pH 8.010x TBE (per litre)108 g Tris base, 55 g boric acid, 40 ml 0.5 M EDTA, pH 8.0 20X TTE buffer (per litre)Tris Base 216 g, Taurine 72 g, EDTA disodium salt 4 g6x gel loading buffer 0.25% Bromophenol blue, 0.25%Xylene cyanol FF, 15% Ficoll Type 4000, 120 mM EDTA