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Method: Plasmid SubcloningMay 20 1990Matthew S. HoltPurpose:The method is used to clone smaller portions of inserts (up to 15 kb in size) which previously have been cloned in YACs phage cosmids or other plasmids.Time required:∙Plasmid Vector Preparation1.Restriction digest and Calf Intestinal Phosphatase (CIP)reaction - 4-6 hours or overnight2.Gel electrophoresis- 4-8 hours or overnight3.Fragment elution and purification - 4-6 hours∙Insert Preparation1.Restriction digest -4-6 hours or overnight2.Gel electrophoresis- 4-8 hours or overnight3.Fragment elution and purification - 4-6 hours∙Ligation Reaction - minimum of 4 hours, best overnightSpecial Reagents:∙10X CIP Buffer∙10X Ligation BufferPreface:Bacterial plasmids are double-stranded circular DNA molecules of 1- 200 kb in size that replicate and are inherited independently of the bacterial chromosome. Different plasmids replicate todifferent extents in their host some reaching a copy number as high as 700 per cell. They function as accessory genetic units frequently containing genes coding for enzymes that are advantageous to the bacterial host. The most commonly conferred phenotype by plasmids is antibiotic resistance though plasmids can also code forantibiotic production restriction and modification enzymescolicins enterotoxins and enzymes used to degrade complex organic compounds. More than one plasmid can occur in a single cell but sincethey compete for the same set of replication enzymes one plasmid usually dominates the others. Over the course of a few generations the minority plasmids are completely eliminated and the descendants of the original cell will contain only one of the original plasmids. Over 30 different "incompatibility" plasmid groups have been identified.Under natural conditions many plasmids are transmitted to a new host by a process known as bacterial conjugation. Newer plasmid vectors however lack the nic/bom site and cannot be conjugated. In the laboratory plasmid DNA can be introduced into modified bacteria (called competent cells) by the process of transformation. Even under the best circumstances plasmids become stably established in a small minority of the bacterial population. Transformants can be easily identified by the selectable marker encoded by the plasmid i.e. antibiotic resistance and the resulting phenotype of those bacterial cells.Uncut plasmid DNA can be in any of five forms - nicked circular linear covalently closed supercoiled or circular single-stranded. When run on a gel one frequently will see these forms as different bands. The exact distances between the bands of these different forms is influenced by percentage of agarose in the gel time of electrophoresis degree of supercoiling and the size of the DNA. One cannot accurately determine size from uncut plasmid DNA. When cut with an enzyme with one recognition site in the plasmid almost all the DNA will fall in one band which equals the linear size of the plasmid (see illustration below).Almost all research plasmid vectors contain a closely arranged series of synthetic restriction sites called the "polylinker". In most cases these restriction sites are unique to the polylinker and hence provide a variety of targets that can be used singly or in combination to clone foreign DNA fragments. The most recent phase of construction of plasmid vectors involves the incorporation ofancillary sequences that are used for a variety of purposes including generating single-stranded DNA templates for DNA sequencing transcription of foreign DNA sequences in vitro and expression of large amounts of foreign protein in vitro.Fragments of foreign DNA can be cloned in a linearized plasmid vector bearing compatible ends by the activity of Bacteriophage T4 DNA Ligase. The enzyme will catalyze the formation of a phosphodiester bond between adjacent nucleotides if one nucleotide contains a 5'-phophate group and the other nucleotide contains a 3'-hydroxyl group.During the ligation reaction however the vector can recircularize without the insert DNA. This can be minimized by removing the5'-phosphate from both termini of the linear vector with Calf Intestinal Alkaline Phosphatase (CIP). Described in the following procedures is a method that has worked consistently. It is recommended to run the vector on a gel cut out the vector band and gel elute to separate the vector from any uncut DNA.The smallest amount of uncut vector will transform efficiently and make finding the bacteria containing the recombinant plasmid virtually impossible. CIP has no effect on uncut circularized plasmids.A fragment of foreign DNA can be inserted into the vector by a process knows as directional cloning by cutting the vector polylinker with two unique restriction sites. Because of the lack of complementary ends the vector fragment cannot circularize efficiently. The insert DNA however must also have the same cohesive termini as the vector. Directional cloning is widely used when insert DNA orientation is specific. Directional cloning also depletes the number of available cloning sites for further constructing. Specific orientations can be obtained by screening several recombinants from a single-enzyme ligation until the desired orientation is identified.Another method of ligation though substantially less efficient is blunt-end ligations. Some restriction enzymes cleave both strands of DNA resulting in blunt ends - i.e. no cohesive overhang. Both the insert and vector termini resulting from any restriction enzyme digest can be made blunt. One method utilizes T4 DNA Polymerase and dNTPs to fill in the overhang. Another method uses the enzyme Mung Bean Nuclease to cleave the single stranded overhang. Ligation reactions involving blunt-ended molecules require much higher concentrations of both vector and insert DNA and T4 DNA ligase. One end blunt ligations are slightly more efficient (and directional)and can be achieved by first blunting the linear DNA and thendigesting with a restriction enzyme unique to only one end of the DNA. Again both vector and insert must have complementary ends.Either of these methods may change the DNA sequence resulting in the loss of the restriction site.When it is impossible to find a suitable match between restrictions sites in the plasmid and those at the ends of the insert synthetic linkers can be ligated to the linearized DNA. The procedure requires blunt-end linear DNA fragments to which ligase adds a series of thesesynthetic restriction sites. By the digesting with that specific restriction enzyme the resulting DNA has only one linker on each end because the DNA-linker bond is not a restriction site.Certain restriction overhangs can be modified using Klenow and the recessed 3' termini can be partially filled to generatecomplementary restriction overhands that are otherwiseincompatible. For instance an insert with BamH1 recognition site G'GATC C C CTAG'G has the overhang GATC after digesting. By filling in with dCTP and dTTP and Klenow the resulting overhand is AG. This is compatible with an Acc1 overhang of AG. Note however that the sequence resulting from this ligation is neither a BamH1 site nor an Acc1 site and an alternative method must be used to cut out the insert.Procedures:Plasmid Vector Preparation1.Digest 10-20 ug of plasmid DNA with the desired restriction enzymein a total volume of 100 ul. Incubate appropriately for yourspecific enzyme (usually 37 degrees C) for 1-4 hours. Supercoiled DNA will take longer to cut and it is recommended to use twice as much enzyme.2.Dephosphorylate the now linear DNA. Add to the completed digest:30 ul 10X CIP Buffer 167 ul ddH2O and 3 ul CIP enzyme (1 unit perul). Incubate at 45 degrees C for 25 minutes.3.Prepare an 0.8-1.2% agarose gel with approximately 6 cm wells. Thiscan be achieved by taping several teeth together. Be certain that the taped comb clears the bottom of the gel bed. Also prepare a mini-gel to run a 5 ul aliquot to verifycomplete digestion anddetermine running time to obtain desired separation.4.Add another 3 ul CIP (probably an unnecessary step by CIPspecification standards) and incubate at 45 degrees C for another25 minutes.5.Inactivate the CIP enzyme by adding 500 ul of phenol. Vortex thenspin in the microfuge at 14000 rpm for 5 minutes. Transfer theaqueous phase to a sterile eppendorf tube.6.Add 500 ul of chloroform (to remove phenol) vortex microfuge at14000 rpm for 2 minutes and transfer the aqueous phase to a sterile tube.7.Precipitate the DNA by adding 30 ul 3M NaAcetate pH 5.2 and 800 ulof -20 degrees C EtOH (must be cold!). Microfuge at 4 degrees Cimmediately for 30 minutes. If you suspect low yields place samples on dry ice for 10 minutes prior to spinning.8.Decant the supernatant immediately and vacuum dessicate or lay tubeon its side to dry the DNA pellet for 15 minutes. Resuspend in 100 ul 1X glycerol dye and load on gel along with a 1kb ladder (BRL) and several molecular weight standards. Run gel 4-8 hours at 80volts until the desired separation is obtained.9.Stain the gel excise band with a clean razor blade and gel elutethe vector (see Gel Elution protocol). Resuspend in 20-50 ul 1X TE and quantitate on a mini-gel. Vector is now ready for ligation.Insert Preparation1.Digest 10-20 ug of insert DNA with the appropriate enzyme in a 100ul volume. Incubate appropriately (usually 37 degrees C) for aminimum of 4 hours. Remove 5 ul for a test gel.2.Prepare an 0.8-1.2% agarose gel with approximately 6 cm wells. Thiscan be achieved by taping several wells together. Be certain that the taped comb clears the bottom of the gel bed. Also prepare amini-gel to run a 5 ul aliquot to verify complete digestion andrunning time to obtain desired separation.3.After verifying complete digestion add 10 ul of 10X glycerol dyeto the digest and load on gel. Run at 80V for 4-8 hours.4.Stain gel excise band(s) with a clean razor blade and gel elute thefragments (see Gel Elution protocol). Resuspend in 20-50 ul 1X TE and quantitate in a mini-gel. Inserts are now ready for ligation.Ligation Reaction1.Maniatis has several complicated formulas to determine the amountsof vector and insert to use for the most efficient ligations. To simplify always have a ratio of 3:1 insert to vector. The more DNA used the more efficient the ligation will be. Insert is usually the limiting factor but try to have between 100-300 ng of vector.2.Set up the reactions with an appropriate amount of vector and insert1 ul of 10X Ligation buffer then adjust the volume to 10 ul withddH2O. Remove 1 ul of the mixture for a negative control then add1 ul of Ligase (2-4 Weiss units per ul) to the original reaction.Incubate the reaction for a minimum of 4 hours though best results are obtained if allowed to ligate overnight (12 hours).Cohesive-end ligations should be done at 11 degrees C blunt-endligations at room temperature.3.Remove another 1 ul of the ligation mixture to test ligationefficiency: Run the aliquot along with the negative control on a mini-gel. The sample with added ligase will have bands of higher molecular weight if a substantial amount of ligation has occurred.e 5 ul of the ligation reaction for transformation (seetransformation procedures). The remaining 4 ul can be stored at -20 degrees C and used as a back-up if necessary.Solutions:1.10X CIP Buffer:2.10 mM ZnCl23.10 mM MgCl24.100 mM Tris pH 8.05.6.10X Ligation Buffer:7.8.0.5 M Tris pH 7.49.0.1 M MgCl210.0.1 M DTT11.10 mM ATP12.10 mM Spermidine13.References:Sambrook J. Fritsch E.F. and T. Maniatis.(1989) Molecular Cloning A Laboratory Manual. Second edition. Cold Spring Harbor Laboratory Press pp.1.53-1.69.。
胶上酶切方法1．切胶：用切胶笔、枪头或刀片切取胶粒（胶粒直径1-2mm），置于1.5ml Appendorf 试管中。
2．清洗：用200ul洗2次，每次10分钟。
3．脱色：对于考染胶，加200ul考染脱色液（25mM NH4HCO3, 50% CH3CN），超声脱色5分钟或37℃20分钟，吸干。
反复洗至胶粒基本无色；对于银染胶，可以不脱色，也可以加100ul银染脱色液(15mM K3Fe(CN)6, 50mM Na2S2O3）轻摇至变为淡黄色透明，再用水反复洗至无色）。
4．脱水：加CH3CN 100ul，使胶粒变白，吸弃CH3CN。
5．还原：加10mM DTT（溶于25mM NH4HCO3）20ul，37℃水浴1小时。
6．烷基化：冷至室温，吸弃溶液。
快速加30mM IAA（溶于25mM NH4HCO3）20ul，置于暗室45min。
（对于二维胶，可以省略还原和烷基化步骤）7．清洗：依次用双蒸水（2次）和50%CH3CN（2次）进行超声或混悬清洗,每次10分钟。
8．脱水：加CH3CN 100ul，使胶粒变白，吸弃CH3CN。
9．将0.1ug/ul酶液用25mM NH4HCO3稀释10-20倍，每管加2-3ul，稍微离心一下，让酶液与胶粒充分接触，4℃放置30min 。
待酶液被胶粒完全吸收后，加入10-15ul 25mM NH4HCO3，37℃过夜。
10. 离心收集酶解液，点靶。
注：1）每加一次溶液，都要旋涡振荡，胶粒要充分浸没在溶液中，进行下一步操作前把溶液吸走。
2）保持溶液和用具洁净，防止污染。
AnchorChip 点样须知1．配基质溶液：0.4mg/ml HCCA in A T (ACN: 0.1%TFA=70:30).2. 点样（二步法）：取样品酶解液1ul (考染样品)或2ul (银染样品)点靶，晾干。
3.点基质：取1ul基质点在晾干的样品上。
4.点标准品：将1 pmol/ul的肽混合物标准品用基质溶液稀释100倍后，取1 ul点靶，晾干。
别人用的protocol.01，切带。
用手术刀切下条带，并切成2～3mm3大小。
2，超纯水涡旋振荡洗2次，10min/次3，考染条带50mM NH4HCO3/ACN 1：1 混合，超声脱色5min或37℃脱色20min，吸去液体。
重复这一步，直至溶液和胶块无色。
银染条带用30mM K3Fe(CN)6和100mM Na2S2O3 1:1 混合，银颜色褪去后水洗至无色。
4，50mM NH4HCO3/ACN 1：1 涡旋振荡洗一次。
5，加100%乙腈(ACN)振荡脱水至胶粒变白，吸去液体，真空干燥5min。
6，加10mM DTT 50ul（1M母液以25mM NH4HCO3稀释）淹没胶块，振荡混匀至胶块泡胀透明。
56℃ 1h.7，冷却至室温，吸干，快速加55mM 碘代乙酰胺（IAM）50ul（淹没胶块），振荡混匀。
（注意快速，避光！）暗处放置45min。
8，25mM NH4HCO3，50mM NH4HCO3/ACN 1：1，100% ACN各洗一次，乙腈ACN脱水至胶粒变白。
真空干燥5min。
9，配制酶反应液。
0.1ug/ul酶储液以25mMNH4HCO3稀释。
考染样品1：10稀释，银染1：20～1：40。
酶与蛋白的质量比约1/40，在1/20~1/100之间都可以。
加入酶液的量以淹没泡胀后的胶体积为宜。
稍离心，让胶块与酶液充分接触，冰上或4℃ 30min。
10，待溶液被胶块充分吸收，吸去多余酶液，加25mM NH4HCO4淹没胶块再多10～20ul，37℃消化过夜。
11，加终浓度1%甲酸（FA）终止反应，振荡混匀，离心。
吸出液相转至新的Ep pendolf管中。
12，胶块用60%ACN/0.1%FA萃取2次，每次15min，吸出液相，合并三次抽提的溶液。
14，溶液真空离心干燥。
0.1%FA水溶液20ul溶解肽段。
-20℃保存待分析。
技术原理详解第一步，脱色。
考染条带用NH4HCO3缓冲液/30%-50%有机溶剂（一般是乙腈）多次水浴超声至胶粒无色，盐离子/有机溶剂的组成同时降低染料和蛋白的盐键和疏水相互作用。
蛋白酶解及质谱鉴定完整操作流程仪器：ABI 4800 MALDI-TOF/TOF串联质谱仪(ABI)、真空冷冻干燥机、水浴锅、Mascot分析软件、移液器、离心管主要溶液配置：考染脱色液：25mmol/L NH4HCO3、50%乙腈的水溶液银染脱色液：15mM K3Fe(CN)6、50mM NaS2O3的水溶液脱水液1：50%的乙腈溶液脱水液2：100%的乙腈吸胀液：25mmol/L NH4HCO3的水溶液酶解覆盖液：25mM的NH4HCO3、10%乙腈的水溶液酶解工作液：含0.02ug/ul胰蛋白酶的酶解覆盖液蛋白萃取液：含5% TFA、67%乙腈的水溶液一、实验操作：1、将0.2ml的枪头前端剪去2cm左右，以增大孔径，2、将枪头垂直戳向凝胶上的蛋白点，旋转枪头直至将胶点取下，3、将取好的胶粒转入装有去离子水的0.5ml离心管里，用枪头反复吸打溶液至枪头内胶粒进入离心管，4、吸干去离子水后封盖保存并做好标记。
二、注意事项：1、离心管最好用进口离心管，以免塑料污染；水最好用去离子水，2、取点时带好口罩与手套以及头套，以免皮屑等角蛋白的污染，3、蛋白鉴定需要的蛋白量越多越好，所以尽可能把某一个点里所有的蛋白全部取到，不过某些比较大的点就只要取一部分就可以了，4、针对特别小的蛋白点，枪头孔径可能会比蛋白点面积大，取点时把该蛋白点的边上空白部分也一起取一些下来也不要紧，5、蛋白点的纯度越高越容易鉴定，一般双向电泳的点都是独立的蛋白，纯度完全满足鉴定的要求，针对有部分交叉的蛋白点，取点时注意不要取混，混合的蛋白点增加质谱鉴定的难度，降低鉴定成功率，6、取完的蛋白点可以放在室温下保存1周左右，可以放在-20度或者-80度保存半年以上，7、只有胶图上清晰可见的蛋白点，其含量就足够用于质谱鉴定，所以只要取一次蛋白点就可以了，不需要把几张胶上的蛋白点取了放在一起进行鉴定，同时注意不要把胶点弄得太碎8、条件允许的话可以多取一次胶点的重复以做好备份。
Goodlett LabIn-gel digestion protocolNote that this protocol requires extreme care in handling the gel slices to avoid contamination with “finger proteins” (skin keratins). The contamination of gel slices with keratins prior to tryptic digestion overwhelms the spectra generated from the tryptic digestion of the protein of interest, preventing identification by MS.At a minimum, scrub/wash all surfaces that will touch the gel with ethanol and then water. Always wear gloves. When removing gloves that you plan to re-use, do not touch the finger areas of the gloves with your bare hands. Use a clean glass surface upon which to lay the gel prior to excision of the band with a new razor blade. Do not allow any part of the gel or the gel slice to touch a surface that may have been touched by your or someone else’s hands.Staining/destaining procedureCoomassie Blue stain: Use Pierce’s Imperial Protein Stain (cat # 24615) andfollow their directions. This is a non-fixing Coomassie Blue stain. After staining, wash the gel many times with at least 100 mL of pure water to remove all SDS(which will interfere with the MS analysis), wash overnight, and several times the next day prior to gel band excision.Silver Staining: see the protocol on the Goodlett Lab website. After development of the gel, wash several times in pure water to remove excess formaldehyde assoon as possible to prevent protein cross-linking. In making up solution C, sodium thiosulphate, make it less than five min prior to use in step 4 or the stainingprocedure will fail.Destaining a Silver Stain: potassium ferricyanide (III) – K3Fe(CN)6; 0.2g in100mLReduction and AlkylationThis procedure is optional, but the idea is to reduce any disulfide bonds in your protein to allow the protease to access the protein better. Alkylation is to prevent the disulfide bonds from reforming.1.Place dry gel pieces in .2mL tubes, add 50-100uL of 20mM DTT (dithiothreitol)in 100mM Ammonium Bicarbonate, and incubate them for 1 h at 60°C2.Remove DTT solution and add 50-100uL of 55mM IAM (iodoacetamide) in100mM ammonium bicarbonate3.Incubate at room temp for 45 min in the dark4.Remove the IAM solution and rinse/vortex the gels for 10 min with 100mMammonium bicarbonate, and then for another 10 min with ACN (acetonitrile).Repeat this procedure.5.Dry the gels using the speed vac (~30 min)Protein digestion1.After excision of the gel slice with a new, clean razor blade, place it in a 1.5 mLEppendorf tube. Add 500 uL of 100 mM ammonium bicarbonate andshake/rotate for 15 min at room temp.2.Discard the liquid and add 500 uL of highly pure acetonitrile (Optima, Fisher, cat# A996-4) and shake/rotate 15 min at room temp. Discard the liquid. Repeat thiswash cycle twice more with ammonium bicarbonate and acetonitrile.3.With a sterile needle, punch several holes in the cap of each Eppendorf tubecontaining a gel slice, close the tube, and Speedvac at room temp for 45 min to remove all liquid from the gel slices.4.To a 20 ug vial of Promega Sequencing Grade trypsin (cat # V511) add 1 mL of50 mM ammonium bicarbonate.5.Add 50 uL to each gel slice and incubate the tube for 45 min on ice. (Do not chopthe gel slice into small pieces since these will be carried over and clog thereverse-phase column used to separate the peptides prior to MS analysis).6.Add enough 50mM ammonium bicarbonate to cover what will be the expandedgel slice and incubate the tube on a shaker (with Scotch tape over the needle holesto prevent evaporation) overnight at room temperature.7.Remove the solution to a new tube in the morning. Add 50 uL of 5%acetonitrile/0.1% TFA to the gel slice and shake 15 min, room temp. Repeat thisstep, pooling each wash with the solution from the overnight digest.8.Finally add 50 uL of 50% acetonitrile/0.1%TFA to the gel slice for 15 min withshaking. Pool with the prior eluates.9.Speedvac the eluates to about 10 uL volume but do not take the peptides todryness since some of the peptides will irreversibly adsorb to the surface of thetube.10.Transfer the solution to an autosampler vial for the mass spectrometer and store at–80C.References:•Shevchekno A, Wilm M, Vorm O, Mann M “Mass-spectrometric sequencing of proteins from silver-stained polyacrylamide gels” Analytical Chemistry,1996 68, 850-858。
