常见实验用溶液的配制方法.doc

常见实验用溶液的配制方法作者：佚名实验频道来源：生物谷点击数： 2985更新时间：2004-12-21 一.常用贮液与溶液1mol/L亚精胺（Spermidine）: 溶解2.55g亚精胺于足量的水中，使终体积为10ml。分装成小份贮存于-20。1mol/L精胺（Spermine）：溶解3.48g精胺于足量的水中，使终体积为10ml。分装成小份贮存于-20。10mol/L乙酸胺（ammonium acetate）：将77.1g乙酸胺溶解于水中，加水定容至1L后，用0.22um孔径的滤膜过滤除菌。10mg/ml牛血清蛋白(BSA）：加100mg的牛血清蛋白（组分V或分子生物学试剂级，无DNA酶）于9.5ml水中（为减少变性，须将蛋白加入水中，而不是将水加入蛋白），盖好盖后，轻轻摇动，直至牛血清蛋白完全溶解为止。不要涡旋混合。加水定容到10ml，然后分装成小份贮存于-20。1mol/L二硫苏糖醇（DTT）：在二硫苏糖醇5g的原装瓶中加32.4ml水，分成小份贮存于-20。或转移100mg的二硫苏糖醇至微量离心管，加0.65ml的水配制成1mol/L二硫苏糖醇溶液。8mol/L乙酸钾（potassium acetate）：溶解78.5g乙酸钾于足量的水中，加水定容到100ml。1mol/L氯化钾（KCl）：溶解7.46g氯化钾于足量的水中，加水定容到100ml。3mol/L乙酸钠（sodium acetate）：溶解40.8g的三水乙酸钠于约90ml水中，用冰乙酸调溶液的pH至5.2，再加水定容到100ml。0.5mol/L EDTA:配制等摩尔的Na2EDTA和NaOH溶液（0.5mol/L），混合后形成EDTA的三钠盐。或称取186.1g的Na2EDTA2H2O和20g的NaOH，并溶于水中，定容至1L。1mol/L HEPES：将23.8gHEPES溶于约90ml的水中，用NaOH调pH（6.8-8.2），然后用水定容至100ml。1mol/L HCl：加8.6ml的浓盐酸至91.4ml的水中。25mg/ml IPGT：溶解250mg的IPGT（异丙基硫代-D-半乳糖苷）于10ml水中，分成小份贮存于-20。1mol/LMgCl2:溶解20.3g MgCl26H2O于足量的水中，定容到100ml。100mmol/L PMSF：溶解174mg的PMSF（苯甲基磺酰氟）于足量的异丙醇中，定容到10ml。分成小份并用铝箔将装液管包裹或贮存于-20。20mg/ml蛋白酶K（proteinase K）：将200mg的蛋白酶L加入到9.5ml水中，轻轻摇动，直至蛋白酶K完全溶解。不要涡旋混合。加水定容到10ml，然后分装成小份贮存于-20。10mg/mlRnase（无DNase）（DNasefree RNase）：溶解10mg的胰蛋白RNA酶于1ml的10mmol/L的乙酸钠水溶液中（pH 5.0）。溶解后于水浴中煮沸15min，使DNA酶失活。用1mol/L的TrisHCl调pH至7.5，于-20贮存。（配制过程中要戴手套）5mol/L氯化钠（NaCl）：溶解29.2g氯化钠于足量的水中，定容至100ml。10N氢氧化钠（NaOH）：溶解400g氢氧化钠颗粒于约0.9L水的烧杯中（磁力搅拌器搅拌），氢氧化钠完全溶解后用水定容至1L。10SDS（十二烷基硫酸钠）：称取100gSDS慢慢转移到约含0.9L的水的烧杯中，用磁力搅拌器搅拌直至完全溶解。用水定容至1L。2mol/L山梨（糖）醇（Sorbitol）：溶解36.4g山梨（糖）醇于足量水中使终体积为100ml。100三氯乙酸（TCA）:在装有500gTCA的试剂瓶中加入100ml水，用磁力搅拌器搅拌直至完全溶解。（稀释液应在临用前配制）2.5 Xgal（5-溴-4-氯-3-吲哚-半乳糖苷）：溶解25mg的Xgal于1ml的二甲基甲酰胺（DMF）,用铝箔包裹装液管，贮存于-20。100Denhardt试剂（Denhardts regent）成分及终浓度配制100ml溶液各成分的用量2%聚蔗糖（Ficoll，400型）2%聚乙烯吡咯烷酮（PVP-40）2%BSA（组分V）水 2g2g2g加水至总体积为100ml依照上表称取各组分，溶于水中定容。过滤除菌及杂质，分装成小份于-20贮存。10标准DNA连接酶缓冲液（standard DNA ligase buffer）（粘端、平端连接）成分及终浓度配制10ml溶液各成分的用量0.5mol/L Tris-HCl100mmol/L MgCl2100mmol/L DTT2mmol/L ATP5mmol/L 盐酸亚精胺（可选）0.5mg/ml BSA（组分V）（可选）水 5ml 1mol/L 贮液1ml 1mol/L 贮液1ml 1mol/L 贮液200ul 100 mmol/L 贮液50ul 1 mmol/L 贮液0.5ml 10 mg/mL 贮液2.25ml将配制好的缓冲液分装成小份，贮存于-20 。100 mmol/L dNTP 溶液（dNTP solutions）可以购买到100mmol/L纯dNTPs贮液，-80可贮存至少6个月。10mmol/L dNTP混合液成分及终浓度配制20ul溶液各成分的用量10mmol/L dATP10mmol/L dCTP 10mmol/L dGTP10mmol/L dTTP水 2ul 100 mmol/L dATP 贮液2ul 100 mmol/L dCTP 贮液2ul 100 mmol/L dGTP 贮液2ul 100 mmol/L dTTP 贮液12ul 20PEG 8000/2.5M NaCl成分及终浓度配制10ml溶液各成分的用量质量浓度为20聚乙二醇2.5mol/L 氯化钠水 20g50ml 5 mol/L 氯化钠 或 14.6g 固体氯化钠补足100ml 加聚乙二醇于含有氯化钠的烧杯中，加水至终体积100ml，用磁力搅拌器搅拌溶解。20SSC成分及终浓度配制1L溶液各成分的用量300mmol/L 柠檬酸三钠（二水）3mol/L 氯化钠水 88.2g175.3g补足1L 溶解柠檬酸三钠（二水）和氯化钠于约0.9L水中，加几滴10N NaOH溶液调pH为7.0，用水补足体积至1L。DEPC（焦碳酸二乙酯）处理水加100ul DEPC 于 100ml 水中，使DEPC的体积分数为0.1。在37温浴至少12h，然后在15 psi 条件下高压灭菌20min，以使残余的DEPC失活。DEPC会与胺起反应，不可用DEPC处理Tris缓冲液。甲酰胺（deionized formamide）直接购买或加Dowex XG8 混合树脂于装有甲酰胺的玻璃烧杯中，用磁力搅拌器轻轻搅拌1h，可去除甲酰胺中的离子。经Whatman 1号滤纸过滤除去树脂后分成小份，充氮气于-80贮存（防止氧化）。磷酸缓冲液（phosphate buffer）按照下表所给定的体积，混合1 mol/L 的磷酸二氢钠（单碱）和1mol/L 磷酸氢二钠（双碱）贮液，获得所需pH的磷酸缓冲液。配制1 mol/L 的磷酸二氢钠（NaH2PO4H2O）贮液：溶解138g于足量水中，使终体积为1L;1mol/L 磷酸氢二钠（Na2HPO4）贮液:溶解142g于足量水中使终体积为1L。1mol/L 磷酸二氢钠（ml）1mol/L 磷酸氢二钠（ml）最终pH值877850815775735685625565510450390330280 123150185225265315375435490550610670720 6.07.07.17.2TE（用于悬浮和贮存DNA）成分及终浓度配制100ml溶液各成分的用量10mmol/L TrisHCl1mmol/L EDTA水 1ml 1mol/L Tris-HCl（pH7.4-8.0，25）200ul 0.5 mol/L EDTA（pH 8.0）98.8ml Tris缓冲液（Tris-HCl buffer）将121g的Tris碱溶解于约0.9L水中，再根据所要求的pH（25下）加一定量的浓盐酸（11.6N），用水调整终体积至1L。浓盐酸的体积（ml）pH8.6142128.53846566671.376 9.08.28.07.2 二.电泳缓冲液、染料和凝胶加样液电泳缓冲液50Tris-乙酸（TAE）缓冲液成分及终浓度配制1L溶液各成分的用量2mol/L Tris碱1mol/L 乙酸100 mmol/L EDTA水 242g57.1 ml的冰乙酸（17.4 mol/L）200ml的0.5 mol/L EDTA（pH 8.0）补足1L 5Tris-硼酸（TBE）缓冲液成分及终浓度配制1L溶液各成分的用量445 mmol/L Tris碱445 mmol/L 硼酸盐10 mmol/L EDTA水 54g27.5g 硼酸20 ml的0.5 mol/L EDTA（pH 8.0）补足1L 染料1溴酚蓝（bromophenol blue）加1g水溶性钠型溴酚蓝于100ml水中，搅拌或涡旋混合直到完全溶解。1二甲苯青FF（xylene cyanole FF）溶解1g二甲苯青FF于足量水中，定容到100ml。10mg/ml的溴化乙锭（ethidium bromide）小心称取1g溴化乙锭，转移到广口瓶中，加100ml水，用磁力搅拌器搅拌直到完全溶解。用铝箔包裹装液管，于4贮存。凝胶上样液（gel loading solutions）6碱性凝胶上样液（室温贮存）成分及终浓度配制10ml溶液各成分用量0.3 N 氢氧化钠6 mmol/L EDTA18聚蔗糖（400型）0.15溴甲酚绿0.25二甲苯青FF水 300ul 10N 氢氧化钠120ul 0.5mol/L EDTA（pH8.0）1.8g15mg25mg补足到10ml 6聚蔗糖凝胶上样液（室温贮存）成分及终浓度配制10ml溶液各成分用量0.15溴酚蓝0.15二甲苯青FF5 mmol/L EDTA15聚蔗糖（400型）水 1.5ml 1溴酚蓝1.5ml 1二甲苯青FF100ul 0.5mol/L EDTA（pH8.0）1.5g补足到10ml 6溴酚蓝/二甲苯青/聚蔗糖凝胶上样液（室温贮存）成分及终浓度配制10ml溶液各成分用量0.25溴酚蓝0.25二甲苯青FF15聚蔗糖（400型）水 2.5ml 1溴酚蓝2.5ml 1二甲苯青FF1.5g 补足到10ml 6甘油凝胶上样液（4贮存）成分及终浓度配制10ml溶液各成分用量0.15溴酚蓝0.15二甲苯青FF5 mmol/L EDTA50甘油水 1.5ml 1溴酚蓝1.5ml 1二甲苯青FF100ul 0.5mol/L EDTA（pH8.0）3ml 3.9ml 6蔗糖凝胶上样液（室温贮存）成分及终浓度配制10ml溶液各成分用量0.15溴酚蓝0.15二甲苯青FF5 mmol/L EDTA40聚蔗糖水 1.5ml 1溴酚蓝1.5ml 1二甲苯青FF100ul 0.5mol/L EDTA（pH8.0）4g 补足到10ml 10十二烷基硫酸钠/甘油凝胶上样液（室温贮存）成分及终浓度配制10ml溶液各成分用量0.2溴酚蓝0.2二甲苯青FF200 mmol/L EDTA0.1SDS50甘油 水 20mg20mg4ml 0.5mol/L EDTA(pH8.0)100ul 10 SDS5ml补足到10ml 三.常用培养基LB培养基将下列组分溶解在0.9L水中：蛋白胨10g酵母提取物5g氯化钠10g如果需要用1N NaOH（1ml）调整pH至7.0，再补足水至1L。注：琼脂平板需添加琼脂粉12g/L,上层琼脂平板添加琼脂粉7g/L。SOB培养基将下列组分溶解在0.9L水中：蛋白胨20g酵母提取物5g氯化钠0.5g1 mol/L 氯化钾2.5ml用水补足体积到1L。分成100ml的小份，高压灭菌。培养基冷却到室温后，再在每100ml的小份中加1ml灭过菌的1mol/L氯化镁。SOC培养基成分、方法同SOB培养基的配制，只是在培养基冷却到室温后，除了在每100ml的小份中加1ml灭过菌的1mol/L氯化镁外，再加2ml灭菌的1mol/L葡萄糖（18g葡萄糖溶于足够水中，再用水补足到100ml，用0.22um的滤膜过滤除菌）。TB培养基将下列组分溶解在0.9L水中：蛋白胨12g酵母提取物24g甘油4ml各组分溶解后高压灭菌。冷却到60，再加100ml灭菌的170mmol/L KH2PO4/0.72 mol/L K2HPO4的溶液（2.31g的KH2PO4和12.54gK2HPO4溶在足量的水中，使终体积为100ml。高压灭菌或用0.22um的滤膜过滤除菌）。2YT培养基将下列组分溶解在0.9L水中：蛋白胨16g酵母提取物10g氯化钠4ml如果需要用1N NaOH（1ml）调整pH至7.0，再补足水至1L。注：琼脂平板需添加琼脂粉12g/L,上层琼脂平板添加琼脂粉7g/L。YPD培养基将下列组分溶解在0.9L水中：蛋白胨20g酵母提取物10g葡萄糖20g用水补足体积为1L后，高压灭菌。建议在高压灭菌之前，对色氨酸营养缺陷型每升培养基添加1.6g色氨酸，因为YPD培养基是色氨酸限制型培养基。为了配制平板，需要在高压灭菌前加入20g琼脂粉。四.常用抗生素氨苄青霉素（ampicillin）（100mg/ml）溶解1g氨苄青霉素钠盐于足量的水中，最后定容至10ml。分装成小份于-20贮存。常以25ug/ml50ug/ml的终浓度添加于生长培养基。羧苄青霉素（carbenicillin）（50mg/ml）溶解0.5g羧苄青霉素二钠盐于足量的水中，最后定容至10ml。分装成小份于-20贮存。常以25ug/ml50ug/ml的终浓度添加于生长培养基。甲氧西林（methicillin）（100mg/ml）溶解1g甲氧西林钠于足量的水中，最后定容至10ml。分装成小份于-20贮存。常以37.5ug/ml终浓度与100ug/ml氨苄青霉素一起添加于生长培养基。卡那霉素（kanamycin）（10mg/ml）溶解100mg卡那霉素于足量的水中，最后定容至10ml。分装成小份于-20贮存。常以10ug/ml50ug/ml的终浓度添加于生长培养基。氯霉素（chloramphenicol）（25mg/ml）溶解250mg氯霉素足量的无水乙醇中，最后定容至10ml。分装成小份于-20贮存。常以12.5ug/ml25ug/ml的终浓度添加于生长培养基。链霉素（streptomycin）（50mg/ml）溶解0.5g链霉素硫酸盐于足量的无水乙醇中，最后定容至10ml。分装成小份于-20贮存。常以10ug/ml50ug/ml的终浓度添加于生长培养基。萘啶酮酸（nalidixic acid）（5mg/ml）溶解50mg萘啶酮酸钠盐于足量的水中，最后定容至10ml。分装成小份于-20贮存。常以15ug/ml的终浓度添加于生长培养基。四环素（tetracyyline）（10mg/ml）溶解100mg四环素盐酸盐于足量的水中，或者将无碱的四环素溶于无水乙醇，定容至10ml。分装成小份用铝箔包裹装液管以免溶液见光，于-20贮存。常以10ug/ml50ug/ml的终浓度添加于生长培养基。蛋白质电泳技术作者：bioinfo实验频道来源：生物谷整理点击数： 8858更新时间：2004-8-6SDS/Polyacrylamide Gel ElectrophoresisStandard SDS-polyacrylamide gel electrophoresis (Laemmli)-gel preparation. Volumes given are sufficient for small (8 cm X 10 cm X 1.5 mm) gel format (10 ml of monomer). Scale up volumes as needed. 1. Pour the Separating GelSet up your gel apparatus, prepare separating gel monomer. Add TEMED just prior to pouring gel (I pour the gels using a pasteur pipet and a rubber bulb). Allow to polymerize before adding stacking gel by overlaying gently with water or n-butanol. With higher % gels, one can immediately pour the stacking gel on the unpolymerized separating gel. Be careful not to mix the two layers. Separating Gels, in 0.375 M Tris, pH 8.87%10%12%15%distilled H2O5.1 ml4.1 ml3.4 ml2.4 ml1.5 M Tris-HCl, pH 8.82.5 ml2.5 ml2.5 ml2.5 ml20% (w/v) SDS0.05 ml0.05 ml0.05 ml0.05 mlAcrylamide/Bis-acrylamide(30%/0.8% w/v)2.3 ml3.3 ml4.0 ml5.0 ml10% (w/v) ammonium persulfate0.05 ml0.05 ml0.05 ml0.05 mlTEMED0.005 ml0.005 ml0.005 ml0.005 mlTotal monomer10.005 ml10.005 ml10.005 ml10.005 ml2. Pour the Stacking GelAfter the separating gel has polymerized, decant the overlay, prepare the stacking monomer, add the TEMED, and pour. Insert the comb and allow to polymerize completely before running. Stacking Gels, 4.0% gel, 0.125 M Tris, pH 6.8distilled H2O3.075 ml0.5 M Tris-HCl, pH 6.81.25 ml20% (w/v) SDS0.025 mlAcrylamide/Bis-acrylamide(30%/0.8% w/v)0.67 ml10% (w/v) ammonium persulfate0.025 mlTEMED0.005 mlTotal Stack monomer5.05 mlFor best results: 1. Make ammonium persulfate solution fresh daily. 2. Degas solutions before adding TEMED for 15 min at room temperature. 3. Running the gelI usually run my gels at constant current, 25-50 mA, depending on gel size. Heres the recipe for 5X SDS-PAGE running buffer. Dilute to 1X before use. 5X Running Buffer, pH 8.3 (1 liter) Tris Base15 g Glycine72 gSDS5 gdistilled water to 1 literStore at room temperature until use. 4. Sample bufferDilute samples at least 1:4 with sample buffer, heat at 95 C for 4 minutes prior to loading. Sample Buffer (8 ml)Distilled water4.0 ml0.5 M Tris-HCl1.0 mlGlycerol0.8 ml10% SDS1.6 mlbeta-mercaptoethanol0.4 ml0.05% (w/v)bromophenol blue0.2 mlSerum Protein Electrophoresis Tricine/Polyacrylamide Gel ElectrophoresisUsed for pilin processing analysis but generally useful for resolution of small (15-35 Kdal) proteins of similar size. SeeStrom, M. S., D. N. Nunn, and S. Lory. 1993. A single bifunctional enzyme, PilD, catalyzes cleavage and N-methylation of proteins belonging to the type IV pilin family. Proc. Natl. Acad. Sci., 90:2404-2408. or Strom, M. S., and S. Lory. 1992. Kinetics and sequence specificity of processing of prepilin by PilD, the Type IV leader peptidase of Pseudomonas aeruginosa. J. Bacteriol. 174:7345-7351. Original citation: Schagger, H. and G. von Jagow. 1987. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166: 368-379. Reagents: Anode Buffer (+): 200 mM Tris pH 8.9 (can dilute from 10X stock) Cathode Buffer (-): 100 mM Tris/100 mM Tricine/0.1% SDS (no need to pH, but will be 8.25) these can be made as 10X stocks and diluted before use Gel Buffer: 3.0 M Tris, pH 8.45/0.3% SDS Note: the pHs given for the anode and gel buffers are essential Stacking acrylamide: 48% acrylamide/1.5% bis-acrylamide Separating acrylamide: 46.5% acrylamide/1.5% bis-acrylamide Sample buffer: (add equal volume to sample), for 20 ml:5 ml 0.5 M Tris, pH 6.8 4 ml 20% SDS 1 ml 2-mercaptoethanol 4 ml 50% glycerol 0.004 g bromophenol blue 6 ml waterPouring Gels For each minigel (Hoeffer Mighty Small or BioRad Mini Protean-II-scale up as required): 1. Separating gel: 15% gel 2 ml separating acrylamide 2 ml gel buffer 2 ml 50% glycerol 10% gel 1.22 ml separating acrylamide 2 ml gel buffer 2 ml 50% glycerol 0.78 ml waterpolymerize with 75 ul 10% APS and 7.5 ul TEMED 2. Stacking gel:0.25 ml stacking acrylamide 0.75 ml gel buffer 2.0 ml water 20 ul 10% APS 2 ul TEMED Polymerize both the stacking and separating gels at the same time (I used small disposable tubes), and pour stacking gel directly onto the separating gel (pour carefully but quickly-they wont mix but the separating gel polymerizes within 2-3 minutes). Make each separating gel mixture separately and add TEMED and APS right before pouring. Multiple stacking gel mixtures can be made in the same tube, but you have around 10 minutes before these start to polymerize too. Sample loading and electrophoresis: For the minigels, 5-10 ul per well gives best results. Layer the samples in the well very carefully, and be sure to flush out any unpolymerized acrylamide before loading. Electrophorese at 25-35 mA (constant current) per gel in minigel setup. Foam will appear on the top (cathode), and additional cathode buffer may have to be added during the run. 15% tricine-PAGESeparation of prepilin and processed pilin from P. aeruginosa on a Coomassie-blue stained tricine-15% PAGE gel. The two forms differ by 6 amino acids after cleavage by1234下一页Rapid ethanol-based Coomassie Blue staining of SDS-polyacrylamide gelsThis protocol was cooked up from a combination of information provided by Novex (electrophoresis products) and by some of my colleagues at NIH, where I was on sabbatical leave last year. The protocol should also work for large format gels (250 x 250 x 1.5 mm gels) as well, but I havent tried it myself. It also works well with methanol-based stain and destain, but significant amounts of methanol vapor are released into the laboratory-the ethanol is safer and just as effective. -Roger Rowlett Procedure for developing 100 x 100 x 1 mm PAGE mingels 1. Place minigel in a loosely covered glass or microwaveable plastic (e.g., tupperware) container, ideally on top of plastic mesh if available. 2. Cover gel with 250 mL of staining solution.* 3. Microwave loosely covered gel/stain on high for approximately 2 minutes or until the solution just begins to boil. (Gels of 10-12% acrylamide are quite robust and will not be damaged even if the solution is boiled for a few minutes.) 4. Place the loosely covered gel/stain container on a slow shaker or simply leave on the bench for 15-60 minutes. It is usually possible to discern bands after as little as 15 minutes. 5. Remove stain from the container (it can be reused many times) and rinse the gel and gel container with water to remove excess staining solution. 6. Cover gel with 200-250 mL of detaining solution (staining solution minus dye). 7. Microwave on high for approximately 2 minutes or until the solution just begins to boil. 8. Place the loosely covered gel/stain container on a slow shaker or simply leave on the bench for 1-24 hours. For rapid destaining, change the destaining solution after one hour and microwave. One to three changes are usually sufficient to visualize bands with a clear background (2-4 hours total destaining time). For overnight destaining, place one large or several small crumpled Kimwipe tissues in the detaining solution to bind up dye, and agitate on a slow shaker. 9. Destained gels are rinsed throroughly with and stored in distilled water. Rinsed gels can be immediately dried in a membrane air-dryer for longer term preservation. * Low-toxicity Staining solution: 0.25 g Coomassie Blue R-250100 mL ethanol100 mL waterStir until dye is completely dissolved, about one hour. Add 25 mL acetic acid and make to 250 mL with water. Store at room temperature in a dark bottle. The final solution is 0.1% Coomassie blue, 10% acetic acid, 40% ethanol. The destaining solution is prepared similarly, but without dye. The original recipe is: 400 mL ethanol100 mL acetic acidmake to 1000 mL with water. Store at room temperature. Bonus tip: 1.0 mm SDS-PAGE minigels run in Tris-glycine buffers can be safely run at 250 V constant voltage (twice the recommended voltage) without any degradation in separation quality. This reduces running time from 90 minutes to about 40 minutes in our hands. Novex claims you can go as high as 300 V, but I havent had the inclination to try that with my homemade gels. Decrease toxicity of destain even more!The destain procedure can be made even less toxic by replacing the destaining solution completely with MilliQ water and no organic solvents. Basically, a freshly stained gel is immersed in water then microwaved on high for 15 min for a 0.75 mm thick gel or 25 min for 1.5 mm thick gel. This method and others are available from Elsevier Trends Technical Tips Online (/tto/index_of_TTO.shtml) under the Electrophoresis section. Reference no. is T01089.Silver Staining of SDS-PAGE gelsIMPORTANT TIPS: DO NOT at any time touch the gel with your fingers.gloved or ungloved.Any contact with the gel with result in a dark stain where the gel was touched. DO NOT use the water from any of the taps.including the deionized water tap. Use only the Milli Q or distilled water. ONLY steps with an * may be allowed to go over the designated times. All other steps should go for only the indicated time. All staining should be done in GLASS (Pyrex) dishes, not plastic trays. It is better to process each gel in a separate dish since they may have different final developing times. The entire protocol may be shortened by reducing incubation times to 20 min each. GEL PREPARATION: 1. Soak gel in SOLUTION #1 for 30 min*-50ml MeOH + 10ml Acetic Acid + 40ml MQ-H2O. Can be made in advance and stored at RT. 2. Soak gel in SOLUTION #2 for 30 min*-5ml MeOH + 7ml Acetic Acid + 88ml MQ-H2O. Can be made in advance and stored at RT. 3. Soak gel in SOLUTION #3 for 30 min -20ml 25% glutaraldyhyde (Sigma G6257) + 30ml MQ-H2O. Make fresh. 4. WASH gel with MQ- H2O either 4 times for 30 min* ( 100 ml/gel/wash) OR 1 time overnight* ( 500 ml/gel/wash). 5. Soak gel for 30 min in DTT solution- 0.5 ml 100x DTT + 50ml MQ-H2O 100X DTT = 0.5 g/ml, store at -20 C GEL STAINING: 1. Soak Gel for 30 min in SILVER SOLUTION - 0.05g Silver Nitrate (Sigma S0139) + 50ml MQ-H2O (be sure to weigh out silver using the analytical balance) 2. Prepare STOP solution- 2.42 g Citric Acid (monohydrate, Sigma C7129) + 5 ml MQ-H2O 3. Prepare DEVELOPER solution-30 ml 37% Formaldehyde (Fisher BP531) + 3g Sodium Carbonate (anhydrous, Sigma S1641)