實(shí)驗(yàn)講義分子生物學(xué)

1、分子生物學(xué)實(shí)驗(yàn)講義實(shí)驗(yàn)一、dna的分離純化-堿裂解法提取質(zhì)粒dna一、 實(shí)驗(yàn)?zāi)康模赫莆諌A裂解法提取質(zhì)粒dna的原理與方法，了解各種試劑的作用。二、 實(shí)驗(yàn)原理：從細(xì)菌分離質(zhì)粒dna的方法都包含3個基本步驟：培養(yǎng)細(xì)菌使質(zhì)粒擴(kuò)增；收集和裂解細(xì)胞；分離和純化質(zhì)粒dna。堿變性法抽提質(zhì)粒是基于染色體dna與質(zhì)粒dna的變性與復(fù)性的差異而達(dá)到分離目的。將細(xì)菌懸浮于葡萄糖等滲液中，經(jīng)edta、naoh- sds溶液處理，可破壞菌體細(xì)胞壁和細(xì)胞膜，使細(xì)胞崩解。naoh破壞核酸堿基對使氫鍵斷裂，細(xì)菌染色體dna和質(zhì)粒dna變性，但質(zhì)粒超螺旋共價閉合環(huán)狀結(jié)構(gòu)的兩條互補(bǔ)鏈不會完全分離，所以當(dāng)以ph4.8的kac高鹽

2、緩沖液調(diào)節(jié)其ph至中性時，變性的質(zhì)粒dna又恢復(fù)到原來的構(gòu)型，重新形成天然的超螺旋分子，并以溶解狀態(tài)存在于液相中。而線狀染色體dna片段難以復(fù)性，與不穩(wěn)定的大分子rna、蛋白質(zhì)-sds-細(xì)胞膜復(fù)合物纏繞附著在細(xì)胞壁碎片上，冰浴后易沉淀，可離心除去。通過氯仿/異戊醇等有機(jī)溶劑除去蛋白質(zhì)，再用乙醇等有機(jī)溶劑沉淀dna，即得純化的質(zhì)粒dna。三、 實(shí)驗(yàn)用品：如試劑: 溶液（葡萄糖維持滲透壓、trishcl維持ph值、edta抑制核酸酶）50mmol/l葡萄糖 25mmol/ltriscl(ph8.0) 10mmol/ledta(ph8.0) 溶液（naoh裂解細(xì)胞與核酸變性、sds裂解細(xì)胞與蛋白質(zhì)變

3、性）0.2mol/lnaoh（臨用前用10mol/l貯存液現(xiàn)用現(xiàn)稀釋） 1%sds 溶液（乙酸鉀置換鈉離子、冰乙酸調(diào)節(jié)ph值）5mol/乙酸鉀60ml 冰乙酸11.5ml 水28.5ml 異丙醇、無水乙醇、蒸餾水四、 實(shí)驗(yàn)步驟：（一）細(xì)菌培養(yǎng)和收集將含有質(zhì)粒的ecoli菌種接種在lb固體培養(yǎng)基（含50 g/ml amp）中，37oc培養(yǎng)1224小時。用無菌牙簽挑取單菌落接種到5ml lb液體培養(yǎng)基（含50g/ml amp）中，37 oc振蕩培養(yǎng)約12小時至對數(shù)生長后期。（二）質(zhì)粒dna的少量快速提?。▔A法）1取1.5ml培養(yǎng)液倒入1.5ml eppendorf管中，12000r/min 離心

4、1min。，棄上清，倒置片刻，使液體流盡。（可重復(fù)步驟1一次，以收集較多菌體）2菌體沉淀重懸于300l溶液中，劇烈振蕩使菌體充分重懸，加入新配制的溶液300l，蓋緊管口，立即將離心管緩慢顛倒數(shù)次直到溶液變清亮 (千萬不要振蕩)。3加入300l預(yù)冷的溶液，蓋緊管口，緩緩顛倒離心管數(shù)次直到白色沉淀充分形成，冰浴5min，12000r/min離心5分鐘。4上清液移入干凈eppendorf管中，加等體積的氯仿/異戊醇（24:1），混勻，放置片刻。12000r/min離心5分鐘。5將水相移入干凈eppendorf管中，加入0.6倍體積異丙醇，震蕩混勻后，12000r/min離心5分鐘。6棄上清液，將管口

5、敞開倒置于衛(wèi)生紙上，使所有液體流出，加入1ml 70%乙醇液沉淀一次，12000r/min離心5分鐘。7吸除上清液，將管倒置于衛(wèi)生紙上使液體流盡，真空干燥10分鐘或室溫干燥。8將沉淀溶于40-50l te緩沖液（ph8.0，含20g/ml rnasea）中，儲于-20冰箱中?！咀⒁狻刻崛∵^程盡量保持低溫。采用酚/氯仿去除蛋白效果較單獨(dú)用酚或氯仿好。沉淀dna通常用冰乙醇，低溫條件下放置時間稍長可使dna沉淀完全。五、實(shí)驗(yàn)結(jié)果：1、記錄觀察現(xiàn)象；2、分析原因如：質(zhì)粒沉淀離心后,在管底能見到少量白色物質(zhì)。真空干燥后，無色，貼于管壁。加ddh2o或te能迅速溶解。質(zhì)粒中含太多雜質(zhì)，則干燥后會呈白色或

6、褐色，不易溶解。實(shí)驗(yàn)二、核酸的檢測（瓊脂糖凝膠電泳）一、 實(shí)驗(yàn)?zāi)康模簩W(xué)習(xí)瓊脂糖凝膠電泳分離dna的原理，掌握dna電泳分析技術(shù)。二、 實(shí)驗(yàn)原理：dna分子帶負(fù)電荷，在電場中受到電荷效應(yīng)、分子篩效應(yīng)向正極移動過程中，因dna分子的大小及構(gòu)象差別而呈現(xiàn)遷移位置上的差異，對于線形dna分子，其電場中的遷移率與其分子量的絕對值成反比。電泳時加溴化乙錠（eb），其與dna結(jié)合形成一種熒光絡(luò)合物，在254-365nm紫外照射下可產(chǎn)生桔紅色的熒光，可用于檢測dna。三、 實(shí)驗(yàn)用品：四、 實(shí)驗(yàn)步驟：1、 瓊脂糖凝膠板的制備： 1g瓊脂糖50ml 電泳緩沖液，加熱熔化均勻，冷卻到6070，加10ul eb 混合

7、，倒入凝膠盒，厚35mm，插上點(diǎn)樣梳，凝固后放入水平電泳槽，使電泳緩沖液淹沒過膠12mm為止。2、 加樣：取出點(diǎn)樣梳，10ul樣品2ul（吸嘴一小滴）上樣緩沖液，混合后取10ul加入點(diǎn)樣孔。同時設(shè)立marker（dna分子量標(biāo)準(zhǔn)物）3、 電泳：6090v，待溴酚藍(lán)移至凝膠的2/3 距離時，停止電泳。4、 凝膠成像系統(tǒng)觀察電泳結(jié)果。五、 實(shí)驗(yàn)結(jié)果：1、 畫出電泳圖。2、 簡單說明分析。如：質(zhì)粒dna在電場中帶負(fù)電向正極移動，用酚、氯仿法從工程菌中提取的質(zhì)粒一般有三種構(gòu)像，即超螺旋，線形和開環(huán)。（開環(huán)質(zhì)粒是指雙鏈環(huán)狀的質(zhì)粒dna有部分解鏈，因此電泳速度最慢）三種構(gòu)像的質(zhì)粒在瓊脂糖電泳的前后順序是超

8、螺旋線形開環(huán)，線形的質(zhì)粒在中間，而開環(huán)的質(zhì)粒在最后。實(shí)驗(yàn)三、聚合酶鏈技術(shù)（pcr鑒定質(zhì)粒）一、實(shí)驗(yàn)?zāi)康模?、熟悉pcr反應(yīng)的基本原理；2、掌握pcr反應(yīng)的操作方法。二、實(shí)驗(yàn)原理：體外快速的dna特異性擴(kuò)增技術(shù)。模擬體內(nèi)dna復(fù)制過程，通過變性、退火、延伸三步反應(yīng)的循環(huán)完成，靶基因的雙鏈dna變性為單鏈，特異的引物在退火過程中與單鏈dna模板聚合，在靶dna的指導(dǎo)下引物的3末端延伸靶dna的互補(bǔ)鏈，完成一個循環(huán)。理論上，每一循環(huán)使dna分子擴(kuò)增一倍，n次循環(huán)后，dna分子擴(kuò)增為2n；高溫變性：94，目的基因變性為單鏈，以作為擴(kuò)增的模板；低溫復(fù)性：5560，一對特異性引物分別與模板3端互補(bǔ)結(jié)合；適

9、溫延伸：72，延伸反應(yīng)；本實(shí)驗(yàn)puc19質(zhì)粒多克隆位點(diǎn)兩側(cè)含有恒定序列，用此序列為特異性引物進(jìn)行pcr擴(kuò)增，產(chǎn)物長度為158bp，以此鑒定puc19質(zhì)粒。三、實(shí)驗(yàn)用品：四、實(shí)驗(yàn)步驟：1、50ul反應(yīng)體系的配制，如表：ddh2o32.6ul10*buffer5ulmgcl2(25mm)5ul待添加的隱藏文字內(nèi)容3dntp(10mm)1ulp1(10um)1ulp2(10um)1ultaq酶(5u/ul)0.4uldna模板4ul 充分混合，放入pcr儀擴(kuò)增3、 反應(yīng)條件：94.0 預(yù)變性 3min 94.0 30s 58.0 20s 30 cycles 72.0 5min 72.0 40s4、2

10、瓊脂糖凝膠電泳檢測（步驟同實(shí)驗(yàn)二）五、實(shí)驗(yàn)結(jié)果：1、畫出電泳圖。2、簡單說明分析。winger tuivasa-sheck, who scored two tries in the kiwis 20-18 semi-final win over england, has been passed fit after a lower-leg injury, while slater has been named at full-back but is still recovering from a knee injury aggravated against usa.both sides boas

11、t 100% records heading into the encounter but australia have not conceded a try since josh charnleys effort in their first pool match against england on the opening day.aussie winger jarryd hayne is the competitions top try scorer with nine, closely followed by tuivasa-sheck with eight.but it is rec

12、ently named rugby league international federation player of the year sonny bill williams who has attracted the most interest in the tournament so far.the kiwi - with a tournament high 17 offloads - has the chance of becoming the first player to win the world cup in both rugby league and rugby union

13、after triumphing with the all blacks in 2011.id give every award back in a heartbeat just to get across the line this weekend, said williams.the (lack of) air up there watch mcayman islands-based webb, the head of fifas anti-racism taskforce, is in london for the football associations 150th annivers

14、ary celebrations and will attend citys premier league match at chelsea on sunday.i am going to be at the match tomorrow and i have asked to meet yaya toure, he told bbc sport.for me its about how he felt and i would like to speak to him first to find out what his experience was.uefa hasopened discip

15、linary proceedings against cskafor the racist behaviour of their fans duringcitys 2-1 win.michel platini, president of european footballs governing body, has also ordered an immediate investigation into the referees actions.cska said they were surprised and disappointed by toures complaint. in a sta

16、tement the russian side added: we found no racist insults from fans of cska. baumgartner the disappointing news: mission aborted.the supersonic descent could happen as early as sunda.the weather plays an important role in this mission. starting at the ground, conditions have to be very calm - winds

17、less than 2 mph, with no precipitation or humidity and limited cloud cover. the balloon, with capsule attached, will move through the lower level of the atmosphere (the troposphere) where our day-to-day weather lives. it will climb higher than the tip of mount everest (5.5 miles/8.85 kilometers), dr

18、ifting even higher than the cruising altitude of commercial airliners (5.6 miles/9.17 kilometers) and into the stratosphere. as he crosses the boundary layer (called the tropopause),e can expect a lot of turbulence.the balloon will slowly drift to the edge of space at 120,000 feet ( then, i would as

19、sume, he will slowly step out onto something resembling an olympic diving platform.they blew it in 2008 when they got caught cold in the final and they will not make the same mistake against the kiwis in manchester.five years ago they cruised through to the final and so far history has repeated itse

20、lf here - the last try they conceded was scored by englands josh charnley in the opening game of the tournament.that could be classed as a weakness, a team under-cooked - but i have been impressed by the kangaroos focus in their games since then.they have been concentrating on the sort of stuff that wins you tough, even contests - strong defence, especially on their own goal-line, completing sets and a good kick-chase. theyve been great