Experiment 4
Agarose Gel Electrophoresis
Electrophoresis is atechnique used to separate and sometimes purify macromolecules-especiallyproteins and nucleic acids, which differ in size, charge or conformation. Assuch, it is one of the most widely used techniques in biochemistry andmolecular biology. When charged molecules are placed in an electric field, theymigrate toward either the positive (anode) or negative (cathode) pole accordingto their charge. In contrast to proteins, which can have either a net positiveor net negative charge, nucleic acids have a consistent negative chargeimparted by their phosphate backbone, and migrate toward the anode.Macromolecules are electrophoresed within a matrix or"gel". According to the difference of materials used to make the gel,gel electrophoresis may be divided into two classes: agaroseand polyacrylamide electrophoresis. Agarose gel electrophoresis is now the most populartechnique used to separate and purify DNA fragments.
Purpose
By the end of this laboratory exercise, youshould be able to describe how to run an agarose gel.
Agarose is a polysaccharideextracted from seaweed. Agarose gels have alarge range of separation, but relatively low resolving power. Byv執(zhí)業(yè)醫(yī)師arying the concentration of agarose, fragments ofDNA from about 200 to 50,000 bp can be separatedusing standard electrophoretic techniques. The higherthe agarose concentration, the "stiffer"the gel. Higher concentrations of agarose facilite separation of small DNAs,while low agarose concentrations allow resolution oflarger DNAs. It is typically used at concentrationsof 0.5 to 2%. The distance DNA has migrated in the gel can be judged byvisually monitoring migration of the tracking dyes. Bromophenolblue and xylene cyanol dyesmigrate through agarose gels at roughly the same rateas double-stranded DNA fragments of 300 and 4000 bp,respectively. When adequate migration has occured, DNA fragments can be visualized by stainingwith ethidium bromide. This fluorescent dyeintercalates between bases of DNA and RNA. It is often incorporated into thegel so that staining occurs during electrophoresis, but the gel can also bestained after electrophoresis by soaking in a dilute solution of ethidium bromide. To visualize DNA or RNA, the gel isplaced on a ultraviolet transilluminator.
Reagentsand apparatus
ü Agarose.
ü TAE(Tris-Acetate,EDTA)[concentratedstocksolution 50X: Trisbase,242g; glacial acetic acid, 57.1ml; 0.5M EDTA (pH8.0),100 ml; add to 600mldH2O, stir vigorously, bring to 1000ml with dH2O].
ü Ethidium bromidestock (10 mg/ml): add 1 g of ethidium bromide to 100ml of H2O, stir with a magnetic stirrer for several hours and trasfer to adark bottle and store at 4℃(Ethidium bromide is a powerful mutagen.Wear gloves and a mask when weighting it out. In case of contact, immediatelyflush with copious amounts of water).
ü TBE [5x stock solution(1 liter): 54 g Tris base, 27.5 g boric acid, 20 ml 0.5 M EDTA, pH 8.0].
ü 10 x loading buffer: 0.25% bromophenolblue, 0.25% xylene cyanolFF, 15% Ficoll in water.
ü Equipment: beaker, graduated cylinder, stirbar, microwave, Pan balace, gel bed, comb,electrophoresis tank, and power supply.
Precedures
Preparation ofthe gel
1. Set upthe gel apparatus as demonstrated. The comb should be straight, and thereshould be a few millimeters of clearance between the bottom of the comb and thebottom of the gel tray.
2. Weigh propoeramount of agarose(e.g.,1.0g agarosein 100ml TAE buffer makes 1% agarose) and dissolve inTAE buffer by heating it in a microwave.
3. Pour the agaroseslowly (aviod bubbles) onto a gel bed with the combinserted. Use the plastic transfer pipette to remove the bubble if there isany. Currently, many varieties of gel beds are available commercially and thosecan be used according to the instruction. However, the “traditional”, as wellas economical and simple gel bed can be made by sealing a proper sized plasticor a glass with the masking tape. Make sure the seal is tight to prevent agarose from leaking, and this can be achieved by runningyour finger along the edge of the gel bed several times.
4. Let gel polymerize for about20 to 60 min depending upon the size of the gel.
5. While the agarose gel is polymerizing, prepare the DNA sample and mixproper amount of DNA with the loading buffer containing the dye such as bromophenolblue.
6. Remove the comb after the agarose gel has polymerized and place the gel bed onto theelectrophoresis tank with the wells near the cathode (black terminal). Fill thetank with proper amount of TAE buffer. Usually the buffer is about 1 cm abovethe gel. Add ethidium bromide (final concentration is1 μg/ml) to the TAE buffer and mixit well. Alternatively, ethidium bromide can be addedto the agarose gel. This can be done by boiling the agarose in the microwave and cooling to about 50℃ and then adding ethidium bromide.
LoadingDNA samples and gel running
7. Hold thepipette perpendicular to the well and add DNA sample slowly with the pipettetip just beneath the opening of the well.
8. After allthe samples have been loaded to the wells, connect the gel tank with the power supplyproperly(black to cathode[-] and red to anode[+]). Set the voltage and timebefore turning on the power supply.
9. Make surethe leads have been properlyconnected by watching the platinum wire at the black terminal near the wells.Bubbles should be slowly rising if the leadshave a good connection.
10. Allowelectrophoresis to progress for appropriate time. The timing of electrophoresisdepends on the length of the gel and the amount of voltage applied. The longerthe gel and the lower the voltage, the longer time is needed. However, highvoltages are significantly less effective at resolving large DNA fragments.
Gel Interpretation
Theuncut DNA lane may have several bands in it. This occurs because國家醫(yī)學考試網(wǎng) the migrationof plasmid DNA in an agarosegel depends on its molecular conformation as well as its size in base pairs. Plasmid DNA can exist in any one of three majorconformations:
Supercoiled -Although a plasmid is usually pictured as an opencircle, within a bacterial cell the DNA strand is coiled around histone-like proteins to form a compact structure. This iscalled supercoiling and this form of the plasmid will move the fastest through the gel due to itscompact structure.
Nicked -During plasmid DNA replication, topoisomeraseI introduces a nick into one strand of the DNA helix and uncoils the plasmid. Physical shearing and enzymatic cleavage during plasmid isolation may also introduce nicks into the supercoiled plasmid to produce arelaxed open circular structure. This form is the slowest migrating form of plasmid. Its “floppy” molecular shape impedes movementthrough the agarose gel.
Linear -Linear plasmid DNA occurs when damage results instrand nicks directly opposite each other on the DNA helix. This DNA moves at arate intermediate between supercoiled and nicked plasmid DNA. The presence of linear DNA in a plasmid preparation is a sign of either nuclease contaminationor sloppy lab procedure.
Gelphotography
DNA fragments separated on the agarosegel after electrophoresis can be visualized and photographed under the UVlight. Many kinds of cameras are available commercially and they can be easilymanipulated following the instructions. In principle, the aperture and thespeed are the two key factors for taking a good picture. For example, toincrease exposure, i.e. to make image more intense, a longer exposure time isrequired and this can be achieved by slowing the shutter speed, or increasingthe aperture. In contrast, to decrease exposure, i.e. to make pictures darker,a shorter exposure time or smaller lens opening is required.
Notes
1. Both TAEand TBE are common buffer used. TBE has relative higher buffering capacity thanTAE.
2. Theloading dye bromophenol blue migrates with DNA about0.5 kb and provides an index of the mobility of the fastest fragments.
3. Themigration of the DNA depends on the following factors:
a) Molecularsize of DNA － thesmaller the DNA, the faster the migration.
b) Agarose concentration – the lower theconcentration, the faster the migration.
c) Conformationof the DNA – circular or nicked DNA often migrate faster: than that of linearDNA.
d) Voltageper cm distance between electrodes – the higher the voltage, the faster themigration.
4. Troubleshooting
If the DNA bands are not sharp and uniform,it may be due to the following reasons:
a) OverloadedDNA
b) Voltagetoo high
c) Torn well
d) Bubble ingel
5. Photographequipment maintenance
實驗四 瓊脂糖凝膠電泳
導言
電泳常用于分離有時也用于純化那些分子大小、電荷性狀或分子構象有所不同的生物大分子－尤其是蛋白質(zhì)和核酸。正因為如此，電泳已成為生物化學和分子生物學中應用最為廣泛的技術之一。當帶電分子置于電場中，根據(jù)它們所帶電荷的不同，不是向正極（陽極)移動，就是向負極（陰極)移動。核酸與蛋白質(zhì)不同，蛋白質(zhì)分子可以帶正電也可以帶負電，而核酸卻只能帶負電，這是由其磷酸鹽分子骨架所決定的，且只能向陽極泳動。生物大分子是在某種基質(zhì)或“凝膠”中遷移的，根據(jù)制備凝膠所用材料的不同，凝膠電泳又分為兩類：瓊脂糖和聚丙烯酰胺凝膠電泳。瓊脂糖凝膠電泳已成為分離和純化DNA片斷應用最普遍的技術。
目的
通過本實驗掌握瓊脂糖凝膠電泳的操作方法。
原理
瓊脂糖是一種海藻多糖，瓊脂糖膠分離范圍很大，但其分辨率卻相對較低。通過改變瓊脂糖凝膠的濃度，應用標準的電泳技術可以分離200 到 50,000 bp大小的DNA片斷。瓊脂糖凝膠濃度越大，凝膠就越硬。較高濃度的瓊脂糖膠有利于較小的DNA的分離，而較低濃度的瓊脂糖膠則可以分離較大的DNA片斷。瓊脂糖膠濃度一般在0.5 到2%之間。通過觀察示蹤染料的遷移距離可以判斷DNA的遷移距離。溴酚藍和二甲苯青染料在瓊脂糖凝膠中的遷移速率大致分別與300和4000 bp大小的雙鏈DNA片斷相同。遷移足夠距離后，就可以通過溴化乙錠染色來觀察DNA片斷。溴化乙錠是一種熒光染料，它嵌插在DNA和RNA堿基之間。它可以在做膠時混入其中在電泳時進行染色，也可以待電泳完成后將凝膠浸泡在稀釋的溴化乙錠溶液中進行染色。必須將凝膠置于紫外分析檢測器中才可以對凝膠中的DNA或RNA進行觀察。
試劑與器材
ü 瓊脂糖
ü TAE（Tris-乙酸，EDTA)[50倍的濃縮儲備液：Tris,242g;冰醋酸57.1ml;0.5M EDTA(pH8.0),100ml;加入600ml蒸餾水，劇烈振搖，再加入蒸餾水至1000ml。]
ü 溴化乙錠儲備液（10mg/ml)：在100ml水中加入1g溴化乙錠，用磁攪拌器攪拌數(shù)小時，轉(zhuǎn)移到黑色瓶中，4℃保存（溴化乙錠是強誘變劑，在稱取時務必帶上手套、面具。一旦接觸到，要立即用大量的水沖洗。)
ü TBE[5倍儲備液（1升)：54gTris堿，27.5g 硼酸，20ml 0.5M EDTA,pH8.0].
ü 10×加樣緩沖液:0.25％溴酚藍,0.25%二甲苯青FF，15％聚蔗糖水溶液。
ü 實驗器材：廣口燒杯，量筒，攪拌棒，微波爐，托盤天平，凝膠板，梳子，電泳槽，電源。
實驗步驟
Ⅰ. 凝膠的制備
1.如圖所示搭好凝膠電泳槽。梳齒應該保持筆直，在梳齒底部與膠槽之間應保持幾毫米的間隙。
2.稱取適量瓊脂糖，加入Tris-乙酸緩沖液中，用微波爐加熱至溶解。（例如稱取1.0g瓊脂糖，溶于100ml Tris-乙酸緩沖液中，制成1％的瓊脂糖)
3. 將瓊脂糖溶液緩緩倒入插有齒梳的凝膠板（避免產(chǎn)生氣泡)。如果有氣泡，用巴斯德吸管去除。目前，許多類型的凝膠板都可以購買得到，按照說明書操作使用即可。而傳統(tǒng)的經(jīng)濟簡便的方法是用膠帶將合適大小的塑料板或玻璃板的邊緣封住，形成一個膠模，以此作為凝膠板�？捎檬种冈谀z板邊緣來回觸摸數(shù)次，確證密封完全，以防瓊脂糖泄漏。
4. 根據(jù)凝膠的大小，讓其凝固大約20至60分鐘。
5.在瓊脂糖凝固過程中，準備好DNA樣品，且向其中加入含有染料如溴酚藍的加樣緩沖液。
6.瓊脂糖凝膠凝固之后，移走齒梳，將凝膠板移至電泳槽上，加樣孔在靠近陰極的一端（黑色端)。向槽中加入適量的TAE緩沖液，通常應沒過膠面1cm。再加入溴化乙錠，混合完全（其最終濃度達到1 μg/ml)。另一種方法是將溴化乙錠加入凝膠中：用微波爐將瓊脂糖煮沸，然后冷卻至50℃，加入溴化乙錠。
Ⅱ.加樣、電泳
7. 用移液槍緩慢將DNA樣品垂直加入加樣孔直至其開口下方。
8. 加完所有樣品后，將電泳槽與電源正確連接（黑色對陰極，紅色對陽極)。打開電源之前要調(diào)好電壓和時間。
9. 觀察鉑絲是否已連接到加樣孔附近的黑色端以確保導線連接正確。如果導線連接正確，會有氣泡緩慢上升。
10. 電泳過程需要一定的時間，其長短取決于凝膠的長度及使用的電壓大小。凝膠越長電壓越低，則所需時間就越長。但是，在分離較大的DNA片斷時，使用高電壓效果不好。
Ⅲ.凝膠（圖象)解釋
未切割質(zhì)粒DNA在其泳道上也許會出現(xiàn)幾個條帶，之所以這樣是由于質(zhì)粒DNA在瓊脂糖凝膠中的遷移距離是由其分子構象及其堿基對大小所決定的。質(zhì)粒DNA可以下列三種主要構象中的任何一種形式存在：
超螺旋－盡管質(zhì)粒通常以開環(huán)的形式進行描述，然而在細菌細胞內(nèi)DNA鏈卻是盤繞在組蛋白樣的蛋白質(zhì)周圍形成一種致密的結(jié)構。這就是所謂超螺旋結(jié)構，由于其結(jié)構致密，它在凝膠中的泳動速度最快。
切口－在質(zhì)粒DNA復制過程中，拓撲異構酶I會在DNA雙螺旋中的一條鏈中引入一個切口，解開質(zhì)粒的超螺旋。在質(zhì)粒分離過程中由于物理剪切和酶的切割作用同樣也會在超螺旋質(zhì)粒中引入切口從而產(chǎn)生松散的開環(huán)結(jié)構。這種形式的質(zhì)粒遷移速率最慢，其“松散”的分子形式阻礙了它在瓊脂糖凝膠中的運動。
線性－當DNA損傷在DNA雙鏈相對應的兩條鏈上同時產(chǎn)生切口時，就會出現(xiàn)線性質(zhì)粒DNA。這種DNA的泳動速率介于超螺旋與切口質(zhì)粒DNA之間。質(zhì)粒制備過程中出現(xiàn)線性DNA說明存在核酸酶污染或?qū)嶒灢僮饔袉栴}。
IV凝膠攝影
電泳之后，在紫外光下可觀察到DNA片斷在瓊脂糖凝膠中得到分離，同時可對凝膠進行拍照。市面上可購買到各種照相機，在說明書的指導下，很容易操作。大體上，要拍好一張照片，照相機的光圈孔徑和感光率是關鍵因素。例如，為了增加曝光度,使影象密度更高，需要加長曝光時間，這可通過減慢快門速度或增加感光率得以實現(xiàn)。與之相反，若要降低曝光度，使影象更模糊，則要縮短曝光時間或減小透鏡孔徑。
注意事項
1.TAE 和 TBE均為常用的緩沖液。TBE比TAE有相對高的緩沖能力。
2. 加樣染料溴酚藍可與長度約為0.5kb的DNA一起遷移，可用于指示遷移率最高的片斷。
3. DNA的遷移速率取決于以下因素:
a)DNA的分子大�。肿恿吭叫�，遷移越快。
b)瓊脂糖濃度－濃度越低，遷移越快。
c) DNA的構象－環(huán)狀的或帶切口環(huán)狀的DNA通常比線狀的DNA遷移要快。
d)兩個電極之間單位厘米的電壓－電壓越高，遷移越快。
4. 問題及原因
如果DNA條帶不夠窄且不夠均勻，可能是由以下原因所引起:
a) DNA過載
b) 電壓過高
c) 加樣孔破損
d) 凝膠中有氣泡
5. 攝影器材的保養(yǎng)
