This shows you the differences between two versions of the page.
— |
horizontalelectrophoresis [25/05/2014 16:46] (current) Consort created |
||
---|---|---|---|
Line 1: | Line 1: | ||
+ | ======Horizontal Gel Electrophoresis ====== | ||
+ | ===== Gel concentration ====== | ||
+ | The range of fragment sizes to be separated will determine the choice of agarose concentration for a gel. Typical agarose concentration is 0.5% to 3.0%. For large DNA fragments low-percentage gels are required, while for small DNA fragments, high-percentage gels are recommended. Weak gels (0.5% agarose) should be electrophoresed at low temperatures (e.g. -4°C). | ||
+ | |||
+ | Agarose gels of 0.75% to 1.0%, for routine electrophoresis, | ||
+ | |||
+ | ===== Electrophoresis buffer ===== | ||
+ | TAE buffer provides optimal resolution of fragments >4 kb in length, while for 0.1 to 3 kb fragments, 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, | ||
+ | |||
+ | ===== Temperature influence ===== | ||
+ | Electrophoresis at high voltages produces heat. Additionally, | ||
+ | |||
+ | ===== RNA mobility ===== | ||
+ | Either before or during electrophoresis, | ||
+ | |||
+ | ===== Separation performance ===== | ||
+ | Gel concentration, | ||
+ | |||
+ | The dye’s fluorescence properties allows the band to be visualised under a UV lamp. However, ethidium bromide may slow the DNA migration rate by approx. 15%. As an alternative, | ||
+ | |||
+ | Note: 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 by soaking the gel for 5 minutes in 0.01 M MgCl2, or for 30 minutes in de-ionised water. Caution! Ethidium bromide is a known mutagen. Always wear gloves when handling. Wear UV safety goggles and protect skin when using any UV light source. | ||
+ | |||
+ | ===== Enhancing resolution ===== | ||
+ | 2 x TAE buffer can be used in units with low buffer volume to enhance resolution during extended runs. | ||