【正文】 純化回收實(shí)驗(yàn)?zāi)康牧私釪NA片段的純化回收基本原理，掌握基本技能實(shí)驗(yàn)原理載體及目的DNA 片斷經(jīng)酶切后，如果無多余的片斷（如載體單切）可以直接酚/氯仿抽提后乙醇沉淀回收直接連接用。實(shí)驗(yàn)步驟In a ml centrifuge tube, add ddH2O μl, 10 buffer μl, 1 mg/ml acetylated BSA μl, μg/μl λDNA μl, 16 U/μl HindⅢ restriction enzyme μl. Incubate at 37℃ for at least 1 h.（加入試劑體積（μl）滅菌水 13，MULT buffer 2，λDNA 4，HindⅢ１，總體積 2０ ）NOTE: Wear rubber or plastic gloves during the following steps. 1 In 250 ml flask, add g agarose and 300 ml TBE (table 1). Melt agarose in microwave oven, mixing several times during heating. Add 2 μl GoldViewTM DNA dye. Cool to about 60℃ keeping covered to avoid evaporation.2 Tape ends of gel tray, insert b, pour agarose in and allow to be solidified.3 Remove tape, place gel tray in electrophoresis tank, fill tank to about 1 mm higher than gel top face with TBE and remove b.4 Heat λDNA (30 ng/μl) and bromophenol blue loading buffer (table 2) mixture at 65℃ for 10 min and place immediately on ice for 5 min. Load in well as molecular weight marker.5 Load samples.6 Run electrophoresis at 125 volt (5 volt/cm) until bromophenol blue has migrated to just above the next set of wells.7 Remove tray from tank and take picture with ultraviolet light in gel image system.注意事項(xiàng) 1 酶切反應(yīng)體系依不同的酶、不同的酶切目的而異。本實(shí)驗(yàn)使用HindⅢ限制性核酸內(nèi)切酶是一類能識別并水解雙鏈DNA中特定堿基順序的核酸水解酶。 雜交背景高可能由于預(yù)雜交時(shí)間短、洗膜不徹底、探針濃高、抗體濃度高和選用錯(cuò)誤類型膜等原因引起。沒用帶正電荷的尼龍膜而用其他類型的膜；探針濃度低或標(biāo)記效率差。 2）用保鮮膜包好，暗室中壓X光片。 5) 用20mL Detection Buffer平衡2～5min。 1) 用50mLWashing buffer短暫浸膜15min。將標(biāo)記好的探針沸水浴變性5－10min，冰上5min，加到雜交袋中，混勻，趕出氣泡?；靹蚝笾?7℃保溫至少1hr，可長至20h以提高標(biāo)記量。 Detection Buffer： TrisHCl、 NaCl及50mmol/L 。本實(shí)驗(yàn)中就是通過連接臂共價(jià)連接在dUTP C11上，如圖13所示。材料Amplify core sequence of Bt gene from genomic DNA of transgenic maize plant and plasmid (CK) with the method of polymerase chain reaction.實(shí)驗(yàn)步驟22. On ice, in ml centrifuge tube, add:StockFor 20 μlFinal concentrationddH2OUp to 20 μl10 buffer μl1 25 mmol/L MgCl2 μl mmol/L mmol/L (each) dNTP μl150 μmol/L1 μmol/L (each) primers μl μmol/LTemplate DNA10 ng/μl pCUSBKHyg ? ng/μl Genomic DNA μl ? μl 5 ng50 ng5 U/μl Taq μl1 U23. Amplify from maize genomic DNA with following program:1 cycle of30 cycles of 1 cycle ofKeeping at94℃ for 5 min94℃ for 30 sec72℃ for 5 min4℃55℃ for 30 sec72℃ for 30 sec24. Amplify from plasmid with following program:1 cycle of30 cycles of 1 cycle ofKeeping at94℃ for 1 min94℃ for 10 sec72℃ for 5 min4℃55℃ for 10 sec72℃ for 30 secNOTE: Wear rubber or plastic gloves during the following steps. 25. In 500 ml flask, add g agarose and 200 ml TBE. Melt agarose in microwave oven, mixing several times during heating. Cool to about 60℃ keeping covered to avoid evaporation.26. Tape ends of gel tray, insert b, pour agarose in and allow to be solidified.27. Remove tape, place gel tray in electrophoresis tank, fill tank to about 1 mm higher than gel top face with TBE and remove b.28. Load Φ174 Hinc II in first well as molecular weight marker and amplified samples one by one.29. Run electrophoresis at 85 volt (5 volt/cm) until bromophenol blue has migrated to just above the next set of wells.30. Remove tray from tank, stain in μg/ml EB for 10 min with gentle shaking.31. Destain in 10 mmol/L MgCl2 for 5 min with gentle shaking.32. Rinse in H2O for 10 min with gentle shaking.33. Take picture with ultraviolet light in gel image system. 實(shí)驗(yàn)六 核酸印跡技術(shù)DIG標(biāo)記探針的分子雜交實(shí)驗(yàn)?zāi)康恼莆諏?shí)驗(yàn)的核酸印跡技術(shù)基本原理，熟悉基本操作實(shí)驗(yàn)原理放射性核素因其靈敏度高而被人們利用在雜交篩選中，但它標(biāo)記探針可利用時(shí)間短（放射性同位素有半衰期，），并且對人體有害，易污染環(huán)境，所以近年來逐步發(fā)展了非放射性雜交篩選。PCR是在試管中進(jìn)行的DNA復(fù)制反應(yīng)，基本原理與細(xì)胞內(nèi)DNA復(fù)制相似，但反應(yīng)體系相對較簡單。它不僅是DNA分析最常用的技術(shù)，而且在DNA重組與表達(dá)、基因結(jié)構(gòu)分析和功能檢測中具有重要的應(yīng)用價(jià)值。實(shí)驗(yàn)五 PCR基因擴(kuò)增及其影響因素分析實(shí)驗(yàn)?zāi)康牧私饩酆厦告準(zhǔn)椒磻?yīng)的原理。DNA經(jīng)溴乙錠（EB）染色，溴乙錠可以插入DNA雙螺旋結(jié)構(gòu)兩個(gè)堿基之間，與核酸形成絡(luò)合物，在紫外（300nm,360nm）激發(fā)下，產(chǎn)生桔黃色熒光（590 nm可見光）。具有不同的相對分子質(zhì)量的DNA片段泳動速度不一樣，可進(jìn)行分離。DNA分子在瓊脂糖凝膠中泳動時(shí)有電荷效應(yīng)和分子篩效應(yīng)。瓊脂糖凝膠電泳也常用于分離、鑒定核酸，如DNA鑒定，DNA限制性內(nèi)切核酸酶圖譜制作等。（4）電泳后區(qū)帶易染色，樣品極易洗脫，便于定量測定。因此，目前多用瓊脂糖為電泳支持物進(jìn)行平板電泳，其優(yōu)點(diǎn)如下。常用的緩沖液有硼酸鹽緩沖液與巴比妥緩沖液。瓊脂糖是從瓊脂中提取出來的， 半乳糖相互結(jié)合的鏈狀多糖。（3）電泳速度快。根據(jù)該原則，植物葉蛋白制備過程中一般需要有四種試劑①離液劑：尿素和硫脲等；②表面活性劑：又稱去垢劑，早期常用NPTritonx100等非離子型去垢劑，離子型去垢劑有SDS、膽酸鈉、LiDS等，還有象CHAPS(含它的蛋白溶液可以凍存)與Zwittergent等雙性離子去垢劑；③還原劑：DTT、DTE、TBP、Trisbase等；④蛋白酶抑制劑及核酸酶：EDTA、PMSF、蛋白酶抑制劑混合物(Proteaseinhibitorcocktails)等，如為了去除緩沖液中存在的痕量重金屬離子，～5mmol/LEDTA，同時(shí)使金屬蛋白酶失活。其特點(diǎn)是： (1)水化作用即蛋白質(zhì)分子表面附有能有效防止蛋白質(zhì)分子沉淀析出的水化膜； (2)電荷排斥作用水化膜外還有電荷層(具陰、陽離子)能有效地防止蛋白質(zhì)分子的凝集。蛋白質(zhì)的制備一般分為以下四個(gè)階段：選擇材料和預(yù)處理，細(xì)胞的破碎及細(xì)胞器的分離，提取和純化，濃細(xì)、干燥和保存。實(shí)驗(yàn)原理以蛋白質(zhì)和結(jié)構(gòu)與功能為基礎(chǔ)，從分子水平上認(rèn)識生命現(xiàn)象，已經(jīng)成為現(xiàn)代生物學(xué)發(fā)展的主要方向，研究蛋白質(zhì)，首先要得到高度純化并具有生物活性的目的物質(zhì)。從含有大量多糖的植物中提取RNA時(shí)應(yīng)在勻漿后離心，并加上以上操作步驟。常見問題分析：得率低：B. RNA沉淀未完全溶解A260/A280 ：，RNA樣品沒有溶于水，而溶于了TE中。2. 勻漿后加氯仿之前樣品可以在60至70℃保存至少一個(gè)月。RNA也可用100℅的去離子甲酰胺溶解，70℃保存。每使用1ml TRIzol至少加1ml 75℅乙醇。每使用1ml ，室溫放置10分鐘。樣品分為三層：底層為黃色有機(jī)相，上層為無色水相和一個(gè)中間層。處理脂肪組織時(shí)，上層有大量油脂應(yīng)去除。一些酵母和細(xì)菌細(xì)胞需用勻漿儀處理。TRIzol的用量應(yīng)根據(jù)培養(yǎng)板面積而定，不取決于細(xì)胞數(shù)。l DEPC處理過的水溶解RNA，儲藏于80℃冰箱備用。材料玉米、水稻、小麥等農(nóng)作物根或葉儀器試劑(一) 儀器 1． 低溫離心機(jī) 2． 分光光度計(jì) 3． 瓊脂糖膠電泳系統(tǒng)，用前先用1% Na OH溶液浸泡過夜，之后再用DEPC水浸泡沖洗。實(shí)驗(yàn)步驟1. Sample leaves (or other organs) from vase or field. It is preferable to use young leaves after darkness treatment of several days, although older leaves that are not senescent may be used.2. Place samples in a vacuum bottle with ice to keep them cool (but do not allow to freeze). Samples may be stored at 80℃ until ready to continue the following steps.NOTE: Once sample frozen, do not allow it to thaw until continuing step 4.3. Remove mid rib, cut leaves into 1 cm sections, put in a mortar prefrozen, add liquid nitrogen, grind to a fine powder with a prefrozen pestle and ladle in a 5 ml centrifuge tube up to about the 2 ml mark. 4. Add 2 ml warm (65℃) CTAB extraction buffer (table 1) and mix several times by gentle inversion.5. Incubate in a 65℃ water bath for 3040 min, mixing gently by inversion every 10 min. NOTE: Wear rubber or plastic gloves and go on with step 69 under a fume hood. 6. Add one volume of chloroform / isoamyl alcohol (24:1) and mi