瓊脂糖凝膠電泳鑒定DNA.doc

實驗十 瓊脂糖凝膠電泳鑒定DNA【實驗?zāi)康摹客ㄟ^實驗掌握瓊脂糖凝膠電泳鑒定DNA的原理與方法?！緦嶒炘怼凯傊鞘菑暮Ｔ逯刑崛〕鰜淼囊环N雜聚多糖，是由D型和L型半乳糖以-1，3和-1，4糖苷鍵相連形成的線狀高聚物（如下圖所示）。瓊脂糖遇冷水膨脹，溶于熱水成溶膠，冷卻后成為孔徑范圍從50nm到大于200nm的凝膠。瓊脂糖凝膠電泳是分離、鑒定和純化DNA片段最為常用的方法之一，這種方法簡便易行。而且瓊脂糖可以灌制成各種形狀、大小和孔徑，在不同的裝置中進行電泳，如果有必要，還能夠從凝膠中回收DNA譜帶。瓊脂糖凝膠的分離范圍較廣，選擇不同凝膠濃度和裝置，從50個堿基對到幾兆不同長度的DNA都可以實現(xiàn)分離。使用電場強度和電泳方向恒定的水平板瓊脂糖凝膠電泳的方法，可以很好的分離長度在50-20,000 bp 的DNA片段。DNA在瓊脂糖凝膠中的遷移率受多種因素影響。例如DNA分子的大小；瓊脂糖的濃度；所加電壓等等。DNA片段越長，泳動速度越慢，而且泳動速度與電場強度成正比。一個給定大小的線性DNA片段，在不同濃度的瓊脂糖凝膠中遷移率不同，DNA電泳遷移率的對數(shù)與凝膠濃度成線性關(guān)系。用低濃度的熒光染料如溴化乙啶染色后，凝膠中的DNA可以直接被檢測出來。紫外燈下可以直接檢測到20pg的雙鏈DNA。【實驗材料】1. 實驗器材水平板電泳槽；灌膠模具及梳齒；電泳儀；55水浴；沸水浴；微量移液器2.實驗試劑（1）DNA樣品；DNA標準分子量標記物；瓊脂糖；1x電泳緩沖液TBE；6x樣品緩沖液（2）溴化乙錠：水中加入溴化乙啶，攪拌數(shù)小時至溶解。將配好的10 mg/ml溴化乙啶溶液裝在棕色瓶中，室溫保存，使用時稀釋至0.5g/ml。緩沖溶液配制表緩沖溶液工作溶液 儲存溶液（每升）TBE0.5x5x0.045mol/L Tris-硼酸54 g Tris 堿0.001mol/L EDTA27.5g 硼酸20ml 0.5mol/L EDTA (pH8.0)6x樣品緩沖液0.2% 溴酚藍儲存溫度450% (w/v) 蔗糖水液【實驗操作】1. 照廠家說明準備好灌膠模具，置于水平面上（實驗操作示意圖如下）。2. 配制1%瓊脂糖凝膠。取250ml三角瓶，加入100ml TBE緩沖液，稱取1克瓊脂糖加入緩沖液中，沸水浴加熱至完全溶解，然后在55水浴中保溫。在灌膠模具中插入梳齒，將稍微冷卻的凝膠倒入灌膠模具。凝膠厚度35mm，避免氣泡產(chǎn)生。3. 室溫放置3045分鐘，待凝膠完全凝固后，按照廠家說明將凝膠放入水平板電泳槽。4. 在電泳槽中加入電泳緩沖液，電泳緩沖液沒過膠面1mm，拔下梳齒形成樣品池。5. 取樣品與6X樣品緩沖液按照比例混合后，用微量移液器緩慢加入樣品池中。6. 蓋上電泳槽并且通電，注意電源正負極，確保樣品向陽極移動。恒壓15V/cm（按照兩極之間距離計算），至示蹤染料溴酚藍前沿距離凝膠前端1cm時，切斷電源。7. 從電泳槽中取出凝膠，將凝膠置于0.5ug/ml溴化乙啶水溶液中，室溫下振搖染色3045分鐘。8. 回收染色液，自來水沖洗凝膠后，于紫外燈下觀察?！咀⒁馐马棥?. 溴化乙啶是一種強誘變劑，并有中度毒性，取用含有這一染料的溶液時務(wù)必戴手套。2. 紫外線對人體，尤其是眼睛有危害性。為減少紫外線照射，必須確保紫外線光源受到遮蔽?！舅伎碱}】影響瓊脂糖凝膠DNA遷移率的因素有那些，分別有怎樣的影響？Experiment 10 Agarose Gel Electrophoresis【Purpose】To master the principle and the method of agarose gel electrophoresis and can use it to identify DNA fragments.【Principle】Agarose is a linear polymer composed of alternating residues of D- and L-galactose joined by -(13) and -(14) glycosidic linkages (as shown in the following figure). Gelation of agarose results in a three dimensional mesh of channels whose diameters range from 50 nm to 200 nm.Agarose Gel Electrophoresis is used to separate, identify, and purify DNA fragments. This technique is simple, rapid to perform. Furthermore, agarose gel can be poured in a variety of shapes, sizes, and porosities and can be run in a number of different configurations. If necessary, these bands of DNA can be recovered from the gel and used for a variety of purposes.Agarose gel has a wide range of separation DNAs from 50 bp to several megabases in length being separated on agarose gels of various concentrations and configurations. Small DNA fragments (50-20,000 bp) are best resolved in agarose gels run in a horizontal configuration in an electric field of constant strength and direction. Several factors determine the rate of migration of DNA through agarose gel, including the molecular size of the DNA, the concentration of agarose, the applied voltage and so on. Larger molecules migrate more slowly because of greater frictional drag and because they worm their way through the pores of the gel less efficiently than smaller molecules. At low voltages the rate of migration of linear DNA fragments is proportional to the voltage applied. A linear DNA fragment of a given size migrates at different rates through gels containing different concentrations of agarose. There is a linear relationship between the logarithm of the electrophoresis mobility of the DNA and the gel concentration.The location of DNA within the gel can be determined directly by staining with low concentrations of fluorescent intercalating dyes, such as ethidium bromide, bands containing as little as 20 pg of double-stranded DNA can be detected by direct examination of the gel in UV. 【Materials】1. Apparatus:（1）Clean, dry horizontal electrophoresis apparatus with chamber and come（2）Boiling water bath（3）Power supply capable of up to 500v and 200mA（4）Water bath preset to 552. Reagent:（1）DNA sample（2）DNA size standards（3）Agarose（4）Ethidium bromide: Add 1 g of ethidium bromide to 100 ml of H2O. Stir on a magnetic stirrer for several hours to ensure that the dye has dissolved. Wrap the container in aluminum foil or transfer the 10 mg/ml solution to a dark bottle and store at room temperatureElectrophoresis buffer bufferWorking solution Stock solution/literTBE0.5x5x0.045mol/L Tris- boracic acid54 g of Tris base0.001mol/L EDTA27.5 boracic acid20 ml of 0.5mol/L EDTA (pH8.0)6xGel-loading buffer0.2% bromophenol blueStorage at 450% (w/v) sucrose in H2O【Procedures】1. Seal the edges of the plastic tray supplied with the electrophoresis apparatus with tape to form a mode (as shown in the figure following). Set the mode on a horizontal section of the bench. 2. Prepare sufficient 1xTBE to fill the electrophoresis tank and to cast the gel.3. Prepare 1% (w/v) agarose solution in electrophoresis buffer: Add 1 g powdered agarose to 100 ml 1xTBE in a 250 ml Erlenmeyer flask.4. Loosely plug the neck of the Erlenmeyer flask with Kimwipes. Heat the slurry in a boiling-water bath until the agarose dissolves.5. Use insulated gloves to transfer the flask into a water bath at 55.6. While the agarose solution is cooling, position the comb 0.5-1.0 mm above the plate so that a complete well is formed when agarose is added to the mode.7. Pour the warm agarose solution into the mode, the gel should be between 3 mm and 5 mm thick. Check that no air bubbles are under or between the teeth of the comb.8. Allow the gel to set completely (30-45 minutes at room temperature), then pour a small amount of electrophoresis buffer on the top of the gel, and carefully remove the comb. Pour off the electrophoresis buffer and carefully remove the tape. Mount the gel in the electrophoresis tank.9. Add just enough electrophoresis buffers to cover the gel to a depth of 1 mm.10. Mix the samples of DNA with 0.2 volumes of the desired 6x gel loading buffer.11. Slowly load the sample mixture into the slots of the submerged gel using a disposable micropipette. Load size standards into slots on both the right and left sides of the gel.12. Close the lid of the gel tank and attach the electrical leads so that the DNA will migrate toward the positive anode (red lead). Apply a