Hi.Today we'll Show you how electro lution is performed in order to purify DNA fragments such as digestion and PCR products. For further purposes like cloning and sequencing, electro lution can be one of the methods of choice. Based on the negative electric charge of DNA, A high cell barrier is placed within the electro field, dropping the fragments and preventing their further migration.
This method allows the purification of a wide range of DNA fragment sizes from different sources with a relatively high efficiency of up to 80%Its low cost. Represent another advantage of electro lution when compared to other purification methods such as columns, resin kits, among others. The DNA sample is first visualized by a tedium bromide stained Aris gel electrophoresis in order to visualize and identified the desired fragment to be purified, electrophoresis should proceed.
Under fragments are well separated, undersized can be precisely determined. Following electrophoresis. Desired fragment is neatly excised from the gel using a brand new razor blade and taking care to turn the gel slice as close as possible to the band of interest.
This procedure should be performed With proper protection from UV light and wearing gloves. After filling the electro luter with the Appropriate burning buffer and taking care to spill it over the surface of the middle plateau, the gel slice is placed inside one of the wheels as close as possible to the V channel. There should be no bubbles inside the vch channel.
This can be avoided by flushing the channel with buffer to remove any trapped air. Then 100 microliters of the high salt barrier are added carefully to the V channel from the site proximal To the well. The chamber is Enclosed and the electrodes connected to the power source to begin electro lution at 100 to 120 volts.
The time lapse will depend on the fragment size for large fragments of up to 20 kbs. 50 minutes to one hour should be enough and small fragments should monitor every 10 minutes with UV light to avoid further elion through the salt solution and into the end adult buffer chain. In this case, we digested 560 nanograms of plasmid with the enzymes ND one and entry to release a 900 base per fragment, a equivalent to 84 nanograms of a sample.
Accordingly, after placing the gel slice inside the well, electro lution will proceed for 25 minutes at 100 volts. Once electro solution is completed and DNA fragments are trapped in the south cushion within the V channel, 400 microliters of solution are recovered from the channel, from the distal side of the receptacle And Placed in a 1.5 microliter micro tube using a fine tip attached to a micro pit, proceed to alcohol precipitation by adding two volumes of cold absolute ethanol and mixed by vortex. Additionally, two to three microliters of glycogen may be added to improve the yield Of DNA recovery classic tubes At minus 20 degrees Celsius for one hour or alternatively, store at this temperature overnight centrifuge at four degrees Celsius at top speed for 25 minutes.
This cardio supernatant and wash by adding one milliliter of cold, 70%ethanol Centrifuge at four degrees Celsius at top speed for five to 10 minutes, discard supernatant and allow to dry it room temperature with the tubes in an inverted position, greases spent in 10 to 15 microliters of nucleus free water and proceed to determine the concentration of the sample. In this case, the purified sample was resuspended in 10 microliters of water and quantified in a spectrophotometer at 260 nanometers wavelength showing a concentration of 6.3 nanograms per microliter, representing a 75%efficiency. You may be thinking you'd be better off investing only 10 minutes of your precious time with a kit.
However, in spite of the time consumed in this process, you will be able to control many of the variables that will allow you to purify a wider range of samples bearing in nature sizes and sources. As you could have seen in the present case, a 75%efficiency was observed equal to or better than most DNA purifying kits. Thank you for joining us today.
We hope that you have found this protocol useful for your research Projects.
