Biochemical experiment Gel extraction & Ligation 4th week

Gel extraction In molecular biology, gel extraction or gel isolation is a technique used to isolate a desired fragment of intact DNA from an agarose gel following agarose gel electrophoresis. After extraction, fragments of interest can be mixed, precipitated, and enzymatically ligated together in several simple steps. This process, usually performed on plasmids, is the basis for rudimentary genetic engineering. After DNA samples are run on an agarose gel, extraction involves four basic steps: identifying the fragments of interest, isolating the corresponding bands, isolating the DNA from those bands, and removing the accompanying salts and stain.

Gel extraction To begin, UV light is shone on the gel in order to illuminate all the ethidium bromide-stained DNA. Care must be taken to avoid exposing the DNA to mutagenic radiation for longer than absolutely necessary. The desired band is identified and physically removed with a cover slip or razor blade. The removed slice of gel should contain the desired DNA inside. An alternative method, utilizing SYBR Safe DNA gel stain and blue-light illumination, avoids the DNA damage associated with ethidium bromide and UV light. Several strategies for isolating and cleaning the DNA fragment of interest exist.

DNA extraction from agarose gels (basic 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. Modern spin-column kits are very good at removing impurities, old-fashioned 'kitchen sink' methods are less good.

DNA extraction from agarose gels (basic method) 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. Place the excised band in a 1.5mL microfuge tube. Three representative methods

Spin-columns (Nucleic acid purification columns) These are excellent for extracting DNA if you can afford them. Dissolve the gel-slice in 3 volumes of chaotropic agent at 50℃ for 10 minutes Apply 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.

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. (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

Dialysis tubing (semi-permeable membrane, Visking tubing) (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 (eg 10µL). The pellet is often so small that it is invisible

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 (eg 3MM paper) to size to fit inside the slit. (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.

Paper strip method (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 (eg 10µL). The pellet is often so small that it is invisible

DNA Ligation Ligation in molecular biology is the joining of two nucleic acid fragments through the action of an enzyme. It is an essential laboratory procedure in the molecular cloning of DNA whereby DNA fragments are joined together to create recombinant DNA molecules, such as when a foreign DNA fragment is inserted into a plasmid. The ends of DNA fragments are joined together by the formation of phosphodiester bonds between the 3'-hydroxyl of one DNA terminus with the 5'- phosphoryl of another. RNA may also be ligated similarly. A co-factor is generally involved in the reaction, and this is usually ATP or NAD+. Ligation in the laboratory is normally performed using T4 DNA ligase, however, procedures for ligation without the use of standard DNA ligase are also popular.

Method Agarose Gel Extraction 1. 정제할 DNA를 포함한 Gel 부분을 잘라낸다. 2. 100mg의 Gel Block을 1.5ml tube에 transfer -> 500ul QGE buffer adding -> Incubation (60℃ , 15 min) Gel이 완전히 녹을 때까지 진행 -> 상온에서 cooling *가끔씩 섞어주면 Gel이 더 잘 녹음 3. 2ml Collection tube에 QGE column 장착 -> step2 의 solution을 spin column에 첨가 -> cfg (13,000rpm, 30sec) 내려간 Solution은 버리고 Spin column 다시 장착 4. Spin column에 W1 buffer 400ul 첨가 -> cfg (13,000rpm, 30sec) 내려간 solution 제거 -> Spin column 다시 장착 -> spin column에 wash buffer 600ul 첨가 후 1분 동안 incubation -> cfg (13,000rpm, 30sec) 내려간 solution 제거 -> Spin column 다시 장착 5. cfg (13,000rpm, 3min - 공회전) -> Collection tube 제거 Spin column을 새로운 1.5 ml micro tube에 장착 6. TDW 30ul 첨가 -> Incubation (Room Temperature, 2min) cfg (13,000rpm, 2min) -> Spin column 제거

Method DNA Ligation 1. Vector , Insert의 양을 정한다. (molar ratio of Insert(3) / Vector(1)) 2. 3. 16℃, overnight 으로 반응시킨다. Vector(9kb) 100ng Insert(0.9kb) 30ng 10X reaction buffer 2ul T4 Ligase 1ul TDW Up to 20ul