A.原理
電泳的高解析力使其成為生化技術中最有效力的一門分析利器，以下簡略說明各種電泳的形式及其 應用。
1.不連續膠體電泳 (disc-PAGE) 是以原態蛋白質進行電泳，一般用作純度檢定或活性分析，SDS- 膠體電泳(SDS-PAGE)則用為次體分子量之測定，梯度(gradient) 電泳則可輔助原態蛋白質分子 量之決定。 結合 SDS 及梯度兩種特性的 SDS-梯度膠體電泳，其解析力最高。
2.電泳形式 早期使用圓柱狀膠體，演變成直立式的平板膠片 (16 × 18 cm)，最後改成迷你電泳 膠片 (8 × 10 cm)，電泳時間只約一小時，而其解析力不變。現在柱狀膠體電泳只用在等電焦 集法，以進行二次元電泳；而大型平板電泳則多用在製備式電泳，一般電泳大多以迷你膠片進 行之。
3.膠體材質 的種類很多，但用在蛋白質者則以 聚丙烯醯胺 (polyacrylamide) 為主；聚丙烯醯胺 電泳是蛋白質應用最廣的電泳分析方式。也有少數使用洋菜膠體 (agarose gel)，多用在測定 pI 較廣的異構脢酵素群。
4. 泳動率：在 pH 8.8 的電泳條件下，大部分 pI 小於 8.8 的分子均能往正極泳動；蛋白質的 泳動率與所帶電荷成正比，而與其分子量成反比；若分子量一樣，但形狀越不規則，或體積越 鬆散者，泳動率越小。
B.儀器用具及藥品 1.迷你電泳槽 2,鑄膠器
3.鑄膠三明治組合：玻璃板 (8x10 cm)、白色氧化鋁板、Spacer 間隔條(0.75 mm) 、Comb 樣本齒 模(0.75 mm)
4.供電器 5.塑膠染色盒 6.旋轉搖盪器
7.試劑組：30% acrylanide mix 、1.5M Tris(pH8.0)、10% Ammonium persulfate、10% SDS 及 TEMED
泡靜置約 20 分鐘。
3.將製膠槽中之 Isopropanol 傾斜倒出，再依序混合表之成分注入製膠槽中，插入樣本齒模以形成 樣品槽。
4.將欲分析之樣品加入 3X loading dye 置於 95℃恆溫器中，加熱 10 分鐘；蛋白質標準液則只需 加熱 5 分鐘。
5.待加熱後之樣品冷卻後，再依序注入 SDS-PAGE 之樣品槽中，並以 110V 進行電泳分析，待藍 色染料色帶跑至膠片底部後即可關閉電源。
6.取出膠片組合，使玻璃板向上，先除去兩側間隔條，再以間隔條輕輕撬起玻璃，膠片則留在 白板上。切除焦集膠體，並在分離膠片的右上方截角做記號 (區別膠片的左右)。
7.將完成之電泳片置於 Staining buffer 中染色約 5 min，染色盤要加密蓋，置於旋轉平台慢速 50 rpm 搖盪之；要注意膠片是否完全沒入染色液中，否則會造成染色不均勻。
8.染色後小心把 CBR 染劑倒回原來瓶中，整塊膠片變成藍色，先用自來水洗掉殘餘的染色液，
再倒入約 20 mL Destaining buffer，加密蓋後再放回旋轉平台搖盪。染色脫色過程請戴手套，
否則會在膠片上留下指紋，而後再以照相系統存取結果即可。
8.離子交換層析分離
A.原理
各種蛋白質分子上可能帶有不同的電性，經過離子交換管柱中的帶電填充膠體，可依其分子帶電性 的差異而分離開來。
B.藥品試劑
1. Buffer A ：50 mM Na3PO4, pH 7.0
2. Buffer B ：50 mM Na3PO4, pH 7.0, 0.5 M NaCl 3. DEAE- cellulose
C.方法步驟
1.震盪 DEAE cellulose 膠體使之懸浮，小心倒入管柱中，讓膠體慢慢沈降，在沈降過程中隨時 加入 buffer A，勿使膠體乾掉。
2.待膠體沈降完全後，高度應在 0.5 cm 左右。膠柱以 buffer A 一直流洗，流速快慢可以不考 慮。
3.樣本的添加方法同上述膠體過濾法，當樣本全部沒入膠體後，收集流出液並以 buffer A 流洗 10 mL 後，去除膠體上方的緩衝液，但勿使膠體乾掉。
4.然後依序以 bufferB 溶離之，流洗約 1 mL。
5.收集衝提液，以進行 SDS-PAGE 電泳分析。
9.金屬層析分離
A.原理
若表現蛋白質上含有一段六個 His 的片段，而親和吸著劑膠體上接有鎳離子，此蛋白質會專一性地 結合到吸著膠體；洗去雜質後可用 imidazole 溶離純質蛋白質
B.藥品配製
1.金屬螯合親和層析膠體
2. Buffer A：50 mM Na3PO4, pH 8.0; 0.3 M NaCl; 20 mM imidazole 3. Buffer B：50 mM Na3PO4, pH 8.0; 0.3 M NaCl; 250 mM imidazole
C.方法步驟
1.親和層析膠體 0.5 mL 已經裝填在管柱內，成為一淡藍色膠柱。請把管柱架直在鐵架上，去除 下方的填塞物，用 buffer A 流洗 20 mL 後塞住出口，準備注入樣本。
2.除去膠面上方的緩衝液，並取出樣本慢慢用滴管加到親和膠體上，小心勿弄亂膠體表面；讓 樣本沒入膠體中。＊收集流出液
3.待樣本全部進入膠體後，關閉出口，再慢慢加入 buffer A，打開出口收集流出液；buffer A 共流洗 10 mL。
4.接著以 buffer B 流洗 1ml。
5.收集衝提液，以進行 SDS-PAGE 電泳分析。
10.酵素活性測定分析
1.取純化後之酵素及粗菌液至於冰水浴中待用。
2.分別依序加入
Reagent Stock Final Volume
H20 675µL
Potassium phosphate buffer, pH8.0 1M 0.1M 100µL D-alanine 0.3M 0.03M 100µL o-phenylenediamine/o-dianisidine 0.3% 0.03% 100µL
Horseraddish peroxidase 0.25U/µL 5U 20µL
Enzyme(DAOase) 5µL
3.將分光光度計開啟，並點選 Time course measurement，在選擇 Measurement 之 parameters 將參 數依下列更改 Photometric mode=abs；Response=quick；wavelength=453nm；End time=120sec；
Data Pitch=0.2sec；Display=auto。
3.利用分光光度計之動力學參數來偵測 OD453 之隨時間變化。
4.分別分析粗菌液、離子交換純化液及金屬層析純化液之間之活性差異。
A Practical
Laboratory Course of Biomolecular
Engineering
Molecular Cloning Techniques
Department of Chemical Engineering National Taiwan University of Science
and Technology
2006/2/13
Table of contents
1 : Polymerase Chain Reaction (PCR) ... 1 2 :Confirmation of PCR Amplification and Initialization of
Expression Plasmid Construction ... 3
3 : Restriction Enzyme Digestion... 5
4 : Expression Plasmid Construction ... 7
5 : Transformation of Escherichia coli by plasmid DNA ... 9
6 : Analysis of Bacterial Growth and Selection of Clones ... 11
7 : DNA Isolation ... 14
8 : SDS-PAGE ... 15
9 : Immuno/Western Blotting ... 18
Molecular Cloning Techniques
The main objective of this laboratory sessions is to introduce graduate students of Chemical Engineering to key techniques utilized in molecular biology and, specifically, molecular cloning. This group of sequential exercises is designed to take students through the natural progressions encountered when inserting a gene of interest in an expression vector for subsequent gene product expression. Students will learn key techniques such as polymerase chain reaction, expression plasmid construction, restriction enzyme digestion and gel electrophoresis as well as acquire useful tools such as bacterial transformation and DNA isolation. These essential applications form the foundation of molecular biology and will provide students with the research tools for examining gene expression in prokaryotic or eukaryotic systems.
Exercise 1: Polymerase Chain Reaction (PCR)
Polymerase Chain reaction (PCR) is a technique used by almost every biology laboratory in the world. This tool is used for the detection of gene products and gene amplification for both research and clinical diagnostic purposes. This technique is very simple and relatively inexpensive requiring two amplification oligonucleotides, a DNA polymerase, a mixture of stabilizing buffers as well as the DNA/RNA template for amplification and analysis. The premise behind PCR is simple, requiring three main phases or steps. In the first phase, the DNA template is denatured at a relatively high temperature (95°C).
The second step requires a lower temperature (50-65°C) for annealing of the oligonucleotides to subsequently amplify the gene/fragment of interest. The final phase is completed at the optimal temperature for polymerase activity (72°C) where the amplified product is elongated from the 3’ end of the primers. The time required for each step is dependent upon the length of the segment to be amplified as well as the source of genetic material (ex: plasmid DNA, genomic DNA, etc.). These steps are repeated in cycles (25-40 cycles/run) in order to amplify sufficient amounts of the product for analysis or applications such as subcloning. PCR has become an essential and reliable tool for basic research as well as clinical diagnostics.
Objective:
Using pEGFP-C1 as a template, students will use PCR to amplify the gene encoding for Enhanced green fluorescent protein (EGFP). The amplified fragment will be visualized using gel electrophoresis and subsequently used to construct a EGFP expression plasmid.
Materials:
pEGFP-C1 ---map on next page PCR Buffers (10X buffer, dNTPs) 5’and 3’EGFP oligonucleotides Taq polymerase
1. Set-up PCR mixture: Students will set-up the PCR mixture (2 x 50 µl) including EGFP-specific oligonucleotides. To one tube, they will add the pEGFP-C1 template DNA. In parallel, they will also set up a negative control tube with water substituted for the DNA template. (Time = 30 minutes)
A) Calculate the amounts of ingredients you will need for each 50µl PCR reaction.
EGFP Rx PCR Components Negative Control Rx
1 µl EGFP Template 0 µl
5 µl 10X buffer 5 µl
1 µl dNTPs 1 µl
1 µl 5’ oligonucleotide 1 µl
1 µl 3’ oligonucleotide 1 µl
1 µl Taq polymerase 1 µl
40 µl water 41 µl
50 µl Total Volume 50 µl
B) Compose a “Master Mix’ including all of the components except for the template and an equivolume of water for the negative control. (Ex: if utilizing 1 µl of DNA template, leave out 1 µl of water from the master mix). Keep the mixtures on ice and add the taq polymerase last.
C) Divide 49 µl of the Master Mix into two PCR reaction tubes on ice.
D) Add 1µl of pEGFP-C1 DNA to one tube. Add 1µl of water to the negative control tube. Close tubes and label accordingly. Keep samples on ice.
2. PCR Amplification: Students will place tubes in the PCR machine, carefully recording their position. They will observe the Lab Assistant set-up the program. The Lab Assistant will review each of the steps of PCR amplification while he/she sets-up the program. Students will be required to record the parameters for their lab report.
The Lab Assistant will begin the amplification. (Time = 20 minutes for set-up and explanation; amplification will take approximately 2-2.5 hours).
Exercise 2: Confirmation of PCR Amplification and Initialization of Expression Plasmid Construction
Objective:
Students will use gel electrophoresis to confirm PCR amplification and begin preparation for expression plasmid construction.
Materials:
Agarose Gel electrophoresis apparatus (trays, combs, boats, power supplies) TAE Buffer (1X)
100 bp DNA Ladder Loading dye
Syber Green I dye
MultiImage Light Cabinet Pipettemen
Pipette tips Eppendorf Tubes Methods:
Preparation of agarose gels:
1.Assemble the minigel electrophoresis apparatus according manufacturer’s instructions.
The BRL Baby Gel is representative of the many gel systems available and comes with a removable through in which tp form the gel. Tape the ends of the trough and position the trough in the apparatus.
2.Melt 0.2g of agarose in 25ml of 1X TAE buffer, using microwave oven or a Bunsen burner, and equilibrate to 45°C in a water bath.
3.fill the trough with agarose and immediately insert the comb. Act quickly to remove any bubbles by touching them with a Pasteur pipet.
4.Allow the gel to solidify at room temperature for 20 minutes. Gently remove the comb.
5.Remove the tape from the trough and fill the reservoirs with 1X TAE buffer to just cover the gel.
Rrunning of the gels:
1. Students will observe the Lab Assistant construct agarose gels for visualization of their amplified PCR products. (Time = 20 minutes)
2. Students will mix 2µl of each of their PCR mixtures with 2µl of diluted loading dye in separate tubes. The remainder of the PCR mixtures will be stored at 4°C for use during expression plasmid construction. (Time = 20 minutes)
A)Set-up two eppendorf tubes in a rack at your bench. Label one tube “EGFP” and the other “-“.
B)Add 2µl of diluted loading dye to each tube.
D)Add 2µl of the negative control PCR reaction to the tube labeled “-”. Mix gently by pipetting.
3. Students will carefully load each of their samples onto the agarose gel. Students must record in which lanes of the gel their samples are loaded. The Lab Assistant will load 100 bp DNA ladder as a marker lane. The gel will undergo electrophoresis for approximately 30-45 minutes. (Time = 1 hour 15 minutes).
4. Students will observe as the Lab Assistant stains the gels and move the gels to MultiImage Light Cabinet for visualization and recording of electrophoresed PCR products. (Time = 20 minutes)
Exercise 3: Restriction Enzyme Digestion
Objective:
In order to get the favorable DNA fragments, Students have to cut the PCR product and pET30b(+) expression plasmid using the same selected restriction enzyme.
Materials:
pET30b(+) expression plasmid ---map on next page EGFP PCR product
1. Assemble the following components in a microcentrifuge tube:
Component Volume
pET30b(+) plasmid 30 µl Restriction enzyme buffer (10x) 5 µl
Restriction enzyme: NdeI 2 µl Restriction enzyme: EcoRI 2µl
water 11 µl
Total Volume 50.0 µl
2. Incubate at the appropriate temperature (usually 37°C) for 6~16 h.
3. Add 7 µl mixtures(loading dye : Syber Green I:water =4:3:3) to the reaction and load the entire sample into a large well on a 1% agarose gel. Run the gel far enough to separate the linear plasmid from nicked and supercoiled species. It is useful to run uncut vector DNA in an adjacent lane to help distinguish undigested from linearized plasmid DNA.
4. Visualize the DNA band with a long wave UV light source and excise the band from the gel using a clean razor blade. Minimize exposure to the light source, which can cause nicks and double strand breaks in the DNA.
5. Recover the DNA from the gel.The Gel extraction DNA Kit is ideal for this application.
Resuspend the final product in a total volume of 35 µl (usually about 50 ng/µl DNA).
Insert preparation:
1. Assemble the following components in a microcentrifuge tube:
Component Volume EGFP PCR product 30 µl Restriction enzyme buffer (10x) 5 µl
Restriction enzyme: NdeI 2 µl Restriction enzyme: EcoRI 2µl
water 11 µl
Total Volume 50.0 µl
2. Incubate at the appropriate temperature (usually 37°C) for 12~16 h.
3. Add 7 µl mixtures(loading dye and Sybr Green I) to the reaction and load the entire sample into a large well (0.5–1.0 cm wide) on a 1% agarose gel. Run the gel far enough to separate the linear plasmid from nicked and supercoiled species. It is useful to run uncut vector DNA in an adjacent lane to help distinguish undigested from linearized plasmid DNA.
4. Visualize the DNA band with a long wave UV light source and excise the band from the gel using a clean razor blade. Minimize exposure to the light source, which can cause nicks and double strand breaks in the DNA.
5. Recover the DNA from the gel.The Gel extraction DNA Kit is ideal for this application.
Resuspend the final product in a total volume of 35 µl (usually about 50 ng/µl DNA).
Exercise 4: Expression Plasmid Construction
Objective:
Students will ligate the PCR-amplified EGFP product with the pET30b(+) expression plasmid. Competent bacteria will be transformed with the plasmid and plated on LB-ampicillin plates for bacterial growth.
Materials:
Spreaders Rotating Plating platforms Top10 competent bacteria
Eppendorf Tubes Ice/ Ice buckets LB Broth Bacterial shaker Eppendorf centrifuges Methods:
1. Students will be supplied with a sample of the pET30b(+) expression plasmid. This plasmid is already digested for PCR fragment insertion and does not require that PCR fragments need to be cleaned up prior to ligation.
2. Ligation of EGFP PCR product with pET30b(+) plasmid: Students will prepare a ligation mix (Total volume = 20 µl) including the pET30b(+) (4 µl) and the EGFP PCR product (12 µl). Add ligase as the last component. Ligation requires 6h at 16°C.
(Time = 7 h) 3. Preparation of LB-Kan Agar plates:
A) Take two LB agar plates to the plate preparation station.
B) Using the pipetteman, add Kan to the surface of your plate. The Kan stock is 30µg/µl.Therefore, add 20µl of the antibiotic to each plate. Be careful to not touch
spreading the ampicillin on each agar plate.
D) Touch the spreader on the agar away from the ampicillin droplet. This ensures that the spreader is no longer too hot. Turn the plate clockwise and you move the spreader back and forth over the surface of the agar to spread the antibiotic evenly.
Repeat for steps C and D for the second plate.
E) Store your plates at 4°C for later use.
4. Bacterial Transformation:
Students will be supplied with a sample of Top10 competent bacteria. Bacteria will be transformed with the ligation mix using the heat-shock method (30 minutes on ice;
120 seconds at 42°C; 2 minutes on ice; add 0.5~1ml of LB broth; 1 hour at 37°C in bacterial shaker). (Time= 1 hour 50 minutes)
A)Pick up a sample of Top10 competent bacteria for bacterial transformation of your pET30b(+)-EGFP plasmid. Keep all components on ice.
B)Add 20µl of the pET30b(+)-EGFP ligation mix prepared in Step 2 to the tube of Top10 competent bacteria. Place this mixture on ice for 30 minutes.
C)After 30 minutes have expired, place the tube containing the mixture in a 42°C water bath for 120 seconds.
D)After 120 seconds, place the mixture back on ice for 2 minutes.
E) Add 0.5~1ml of LB broth to the mixture and place the tube at 37°C in the bacterial shaker for 1 hour.
F)Retrieve the LB-Kan plated from the 4°C refrigerator to warm up to room
F)Retrieve the LB-Kan plated from the 4°C refrigerator to warm up to room