Helpful Tips for Horizontal Electrophoresis

We have compiled a general list of helpful tips for using horiztonal gel electrophoresis units


Casting Tip: Regardless of the casting method, esure leak-free casting by lining the casting unit with agarose and allowing it to set before pouring the remainder of the hand-hot agarose mixture i.e at approx 50ºC

Technical Tip: Gasket too tight or loose? Simply remove the gaskets from the ends of the gel trays and refit them into the groove. Refit with gasket protruding slightly from the ends if the gel tray was too tight, or with the gasket fitted below the top edges if the gasket was too loose.

Technical Tip: DNA Mobility: DNA fragments as small as 1kb or less can be separated using agarose gel electrophoresis. For fragments smaller than 0.1kb, polyacrylamide gels are more suitable.

Technical Tip: RNA Mobility: Either before or during electrophoresis, RNA should be denatured. For example:
a.) RNA fragments which have been denatured with glyoxal and dimethyl sulphoxide can be separated on neutral agarose gels
b.)RNA can be fractionated on agarose gels containing methylmercuric hydroxide or formaldehyde.
c.) RNA samples usually require longer runs or buffers that are easily depleted, so it is necessary to circulate the buffer. Northern analyses should not be run on a mini-gel tank.

Technical Tip: Electrophoresis Buffer Selection: TAE buffer provides optimal resolution of fragments >4kb in length, while for 0.1 to 3kb fragments the TBE buffer should be selected. TBE has both a higher buffering capacity and lower conductivity than TAE and therefore should be used for high-voltage electrophoresis. Additionally, TBE buffer generates less heat than TAE at an equivalent voltage and does not allow a significant pH drift.

Technical Tip: Temperature: Electrophoresis at high voltages produces heat. Additionally, high-conductivity buffers such as TAE generate more heat than low-conductivity buffers. Care should be taken in agarose gel electrophoresis with voltages greater than 175 volts, as heat build up can generate gel artefacts such as S-shaped migration fronts, and in extended electrophoresis runs can even melt the agarose gel. With high voltage electrophoresis, the use of low-melting point agarose gels should be avoided.

Technical Tip: Separation Performance: Gel concentration, running buffer, voltage, temperature, nucleic acid conformation and the presence of ethidium bromide all affect separation results. To establish the progress of double stranded DNA, ethidium bromide (0.5 ug/ml) is often added to the running buffer. The dye's fluorescent properties allow the bands to be visualized under a UV lamp. However, ethidium bromide may slow down the DNA migration rate by approx 15%. As an alternative, after electrophoresis, the gel may be stained in and ethidium bromide solution (0.5ug/ml H20) for 15 to 60 minutes and then viewed or photographed on a UV transilluminator. Note: the staining time should be minimised to prevent small nucleic acid fragments from diffusing out of the gel. Background fluorescence of unbound ethidium bromide can be minimised through destaining (Soaking the gel for 5 minutes in 0.01M MgCl2 or for 30 minutes in de-ionised water.

Technical Tip: Ethidium bromide is a known mutagen. Always wear gloves when handling. Wear UV safety goggles and protect skin when using any UV light source.

Technical Tip: Photography: If the gel needs to be photographed, thin gels (2 - 3mm) with low-percentage agarose are better than thick or high-percentage gels. The latter produce increaded opaqueness and autofluorescence.

General Tip: We recommend 0.67ml of agarose per cm² of gel tray area for a minimum 5mm thick gel- e.g. 150ml og mrlyrf shstodr got s 15x15cm gel tray. (Volume ml = W x L x 0.67)



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