1 / 7

DNA extraction from agarose gels (basic method)

DNA extraction from agarose gels (basic method). Kris-itd.unair.ac.id (for education purpose only.

kenneth-douglas
Download Presentation

DNA extraction from agarose gels (basic method)

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. DNA extraction from agarose gels (basic method) Kris-itd.unair.ac.id (for education purpose only

  2. Which method ? There are many different methods of extracting DNA bands from an agarose gel. Which one you choose will probably depend on the consumables you have available in your lab. Another important consideration is the yield/purity of the DNA after extraction. Agarose contains various impurities which may inhibit downstream reactions if not efficiently removed from the DNA. We used to give the generic term 'hoonose' to the unspecified carbohydrates that co-purified along with the DNA and inhibited enzyme reactions. Modern spin-column kits are very good at removing impurities, old-fashioned 'kitchen sink' methods are less good.

  3. Cutting out the DNA band Most of the methods require you to cut out the band. You must visualise the band, in an ethidium bromide stained gel, in a dark-room on a UV light-box (a trans-illuminator). UV is dangerous, wear gloves, long-sleeves and face protection. Never look at UV with unprotected eyes. If possible, set the trans-illuminator to long-wavelegth UV (or low-power) and minimise the amount of time the DNA is exposed. This is because the UV mutagenises the DNA at a measurable rate. Use a scalpel blade to cut around the band of interest. Switch off the transilluminator, switch on the white light and carefully remove the band from the gel and place it on the glass. It is good to trim off as much empty agarose as possible so go back to UV illumination briefly to do this. Place the excised band in a 1.5mL microfuge tube.

  4. Three representative methods : 1 . Spin-columns (Nucleic acid purification columns) 2. Dialysis tubing (semi-permeable membrane, Visking tubing) 3. Paper strip method

  5. Spin-columns (Nucleic acid purification columns) These are excellent for extracting DNA if you can afford them. They cost 1–2 US$ each. Manufacterers include Qiagen, Sigma, Novagen. Everything you need comes with the kit, including protocol, but to summarise; Dissolve the gel-slice in 3 volumes of chaotropic agent at 50C for 10 minutesApply the solution to a spin-column and spin for 1 minute (the DNA remains in the column)Wash the column by passing 70% ethanol through (the DNA remains in the column, salt and impurities are washed out)Elute the DNA in a small volume (30µL) of water or buffer, spin to collect.

  6. Dialysis tubing (semi-permeable membrane, Visking tubing)(1) Freeze the gel slice at –20C for 30 minutes. This is to make it easier to handle the gel slice(2) Cut a 5cm length of dialysis tubing and rinse it inside and out with distilled water. Then rinse it with the same buffer used for the gel (eg. 0.5 x TBE) and leave it submerged in a small beaker of this buffer. Seal one end with a dialysis clip Dialysis tubing (Sigma D9777) is purchased in rolls (dried), prepared by boiling and stored submerged in buffer at 4C. Dialysis clips are Sigma Z37,096-7.(3) Insert the frozen gel-slice into the tubing and add 200–400µL of buffer (eg. 0.5 x TBE). Seal the other end of the tubing with a second dialysis clip. The buffer around the gel-slice must be the same as the buffer inside the gel(4) Immerse the sealed tubing in an electrophoresis tank so that the DNA band is parallel to the electrodes and apply 5V/cm electric field. The DNA will migrate out if the gel towards the positive electrode. It will be retained by the dialysis tubing. You can see this happening under long-wavelength UV if you like. It takes about 10–15 minutes.(5) Remove the buffer from the tubing and place into a 1.5mL microfuge tube.(6) Phenol/chloroform extract and ethanol precipitate the DNA. Re-dissolve the DNA pellet in an appropriate volume of water or TE buffer (e.g. 10µL). The pellet is often so small that it is invisible

  7. Paper strip method(1) Using a scalpel blade, cut a slit immediately in front of the band to be extracted. Do not remove the band from the gel(2) Cut a piece of filter paper (e.g. 3MM paper) to size to fit inside the slit. For example, 3mm x 10mm(3) Place the paper strip in the slit, return the gel to the electrophoresis tank (submerged in buffer) and switch on the current for 2–5 minutes. The DNA runs onward into the paper and is delayed in the smaller mesh size of the paper. Eventually the DNA will pass through so you have to keep checking it under long-wavelength UV so as not to leave it too long.(4) Remove the strip of paper (carrying at least some of the DNA) and place into a 0.5ml microfuge tube, DNA side down.(5) Make a tiny hole in the bottom of the tube using a needle (CAREFUL!)(6) Place the 0.5mL tube inside a 1.5mL tube and spin for 30 seconds. You may have to remove the lid of the 1.5mL tube. The buffer and DNA are retained in the larger tube. You can add 100µL of TE to the paper and re-spin to get a little more DNA out.(7) Phenol/chloroform extract and ethanol precipitate the DNA. Re-dissolve the DNA pellet in an appropriate volume of water or TE buffer (e.g. 10µL). The pellet is often so small that it is invisible

More Related