分子生物学相关技术.doc

一般市售乙醇有两种,一种是无水乙醇(99.0%以上),一种是95%乙醇,通常称为九五醇.这里的100%或者95%一般是指质量百分数.不知道你的90%是质量百分数还是体积百分数.以无水乙醇为例：1、体积百分数。举例说明，如果你要配制1000ml90%乙醇，手上有无水乙醇，假设需要Yml无水乙醇来配制，那么，Y=1000*90%.水的体积就是1000-Y。2、质量百分数。举例说明，如果你要配制1000ml90%的乙醇，手上有无水乙醇，无水乙醇的密度为a,假设需要Yml无水乙醇来配制，那么，Y=1000*90%/a。水的体积就是1000-Y。楼上两位说得对，一般都应该用蒸馏水来配置。MOPS学名：3-(N-Morpholino)propanesulfonic acid中文名：3-(N-吗啉基）丙磺酸分子式：C7H15NO4SpH 范围：6.5 - 7.9，pKa (25) ：7.2。用途：适用于叶绿体薄层备样的电子传递和磷酸化研究；可以配制成多种琼脂培养基，在极限培养基中作为无毒缓冲液应用于棒状链霉菌（Streptomyces clavuligerus）的培养和头孢菌素（cephalosporin）的生产；可作为二维凝胶电泳中等电聚焦电泳（IEF）的电解质系统成分；还可应用于Northern杂交，作为RNA的分离和转膜时的缓冲液。使用方法：直接添加至无菌培养基或在配置培养基时加入一起过滤除菌使用。储存条件：4保存。新加坡普式现货价格(Mean Of Platts Singapore)是新加坡普式公司按照市场的燃料油供需情况来定下的一个独立于其他公司的价格，以供市场参考。进口燃料油的一般成本=(MOPS价格+到岸贴水)汇率1.2402+其他各项费用(1)Current address:/liscum/LiscumLabPage.html(site contains updated information)Division of Biological Sciences105 Tucker HallUniversity of MissouriColumbia, MO 65211phone: 573-882-2672fax: 573-882-0123email: (2)Current address:DNA Plant Technology Corporation6701 San Pablo AvenueOakland, CA 94609phone: 510-547-2395fax: 510-547-2817email: NOTE: The protocol presented below is based on the Amplified Fragment Length Polymorphism (AFLP) technology developed by Marc Zabeau and colleagues at Keygene N.V., Agrobusiness Park 90, P.O. Box 216, NL-6700 AE Wageningen, Netherlands (Zabeau, 1992; Zabeau and Vos, 1993; Vos et al., 1995). The AFLP technology is covered by patents and patent applications owned by Keygene N.V. Both, Life Technologies (Gathersberg, MD, USA) and Perkin Elmer (Applied Biosystems Division, Foster City, CA, USA) market research kits (under license) for AFLP fingerprinting of plant DNAs.Background RationalAFLP as developed by Keygene, was designed as a highly sensitive method for DNA fingerprinting to be used in a variety of fields, including plant and animal breeding, medical diagnostics, forensic analysis and microbial typing, to name a few. We are using this technology to generate DNA based markers for cloning genes involved in phototropic responses in higher plants that have only been identified genetically by mutant phenotype(s) (see Liscum and Briggs, 1995). We have had tremendous success to date using the technology decribed below (see pg. 4). (Recently, Thomas et al. (1995) reported the use of AFLP technology in the identification of tightly linked markers flanking (within 15.5 Kb) the Cf-9 resistance gene of tomato. Although the Cf-9 gene had been previously isolated via transposon tagging the rapid identification of markers within 15.5 Kb of the locus provide support for the contention that AFLP technology can be exploited for gene isolation by positional cloning.) The guts of how AFLP works is summarized in the following paragraphs.The power of AFLP is based upon the molecular genetic variations that exist between closely related species, varieties or cultivars. These variations in DNA sequence are exploited by the AFLP technology such that fingerprints of particular genotypes can be routinely generated. These fingerprints are simply RFLPs visualized by selective PCR amplification of DNA restriction fragments. In order to give a brief summary of the working theory behind AFLP we will describe the technique as we use it to generate AFLPs between two Arabidopsis ecotypes, Columbia and Landsberg erecta. 1) Genomic DNA from each ecotype is digested to completion with two restriction enzymes, one EcoRI (having a 6 bp recognition site) and the other MseI (having a 4 bp recognition site). EcoRI cuts every 2-2.5 Kb in Arabidopsis, while MseI cuts every 300-400 bp. Thus a large number of fragments are generated with relative abundances as such: MseI-MseI MseI-EcoRI EcoRI-EcoRI. As discussed below we target the MseI-EcoRI and EcoRI-EcoRI fragments for analysis. 2) Specific ds oligonucleotide adapters ( 25-30 bp) are ligated to the restricted DNA fragments. 3) Oligos homologous to the adapters, but having extensions at the 3-end are used to amplify a subset of the DNA fragments. These extensions can vary in length from 1 to 3 bp, but are of defined length for a given primer. The sequence of the extension can also vary from one primer to another but is of a single, defined sequence within a given primer. The selective nature of AFLP-PCR is based on the 3 extensions on the oligonucleotide primers. Since these extensions are not homologous to adapter sequence, only plant DNA fragments complementary to the extensions will be amplified due to the inability of Taq DNA polymerase, unlike some other DNA polymerases, to extend DNAs if mismatches occur at the 3-end of a molecule that is being synthesized. Therefore only a subset of the entire genome is amplified in any reaction. For example, if 2 bp extensions are used only one in 256 molecules is amplified assuming Taq polymerase cannot tolerate mismatches as discussed above. However, since MseI-MseI fragments predominate the population of fragments to be amplified, we need to further limit the number of fragments that are actually visualized so that a manageable number is observed. We do this by labeling the primer directed against the EcoRI-adapter sequence since MseI-EcoRI and EcoRI-EcoRI fragments will be a more limiting subset of the total DNA fragments. Finally, the amplified DNAs are separated on a polyacrylamide gel (sequencing type) and an autoradiograph is generated.By labeling the EcoRI-directed primer and using 2 bp extensions on both primers we typically observed 100-200 bands on the autorad from any given primer pair. Only a subset of these total bands are polymorphic between two related individuals, such as Arabidopsis Columbia and Landsberg erecta ecotypes. Any given AFLP primer pair generates on average 10-20 ecotype specific bands (conservatively), thus given 256 possible primer pair combinations when using 2 bp selective 3 ends, 2500-5000 markers can be generated quite rapidly. Given a genome size of 100 Mbp for Arabidopsis, and assuming equal recombination frequencies over the entire genome (or equal distribution of markers throughout the genome), generation of 2500 markers would place any pair of markers within 40 Kb of each other, on average. This level of physical linkage would result is the generation of markers that are _ 0.3 cM (0.3% recombination) within the mutant locus of interest. Thus AFLP should eliminate the walk typically associated with non-T-DNA-tagged mutants, and substitute the hop or land.AFLP Protocol (Abridged Version 1.3, 12/95)1.0 Generate polymorphic recombinant F2 (or F3) populationOutcross desired mutant in one genetic background (parental ecotype of the mutant hence referred to as WT1) to a wild-type plant of another genetic background (WT2) known to be molecular genetically polymorphic to WT1. We have used three WT ecotypes of Arabidopsis for our work (Columbia, Landsberg erecta, & WS) and each is about equally polymorphic from any other. There is no reason to believe that other ecotypes would not be equally suitable. Self the resultant F1 seed and select homozygous mutants from the subsequent F2 generation. Collect tissue from homozygous mutant F2 plants, as well as WT1 and WT2 plants, for DNA isolation as described below inSec. 1.1. Self homozygous mutant F2s and collect seed for future use. It is also helpful to save a few non-mutant F2s for tissue collection and F3 seed collection as they may be useful controls for future experiments.1.1 Isolate genomic DNANOTE: Use any mini(micro)prep that yields good quality DNA. CsCl-purified DNA is not necessary. The main contaminants of concern are carbohydrates. If the A260/A230 is _ 2.2 the nucleic acid should be of adequate quality. Dark adapting the plants for 2-3 d prior to tissue harvest is usually enough to insure low carbohydrate content.Microprep1.) Dark adapt 3-4 week old plants (just beginning to bolt) for 2-3 d, then harvest tissue (1-4 rosette leaves) into 1.5 mL microfuge tubes w/ liquid N2. Grind tissue on liquid N2 with pellet pestle (Kontes Scientific Glassware/Instruments; cat #749520-9001 for pestles only, #749520-0000 for tubes and pestles). Using an electric drill to hold and rotate the pestle works extremely well! Hold ground tissue at _ -80 C until extraction.2.) For extraction of nucleic acids, add 750 L extraction buffer (see below) to ground tissue, vortex to mix thoroughly, and incubate 65 C for 10 min.Extraction Buffer50 mM Tris, pH 8.010 mM EDTA, pH 8.0100 mM NaCl1.0 % (w/v) SDS10 mM -mercaptoethanol (add just before use)3) Add 150 L 5 M K-acetate, vortex, incubate on ice for 20 min. Spin 12K x g in microfuge for 10 min.4) Transfer 750-800 L supernatant to new microfuge tube and add an equal vol of isopropanol. Immediately spin down nucleic acids by centrifugation 12K x g for 1 min. Some small pieces of tissue may pellet at this stage, but they will be removed in the next step.5) Wash pellet w/ 75% EtOH and resuspend in 200 L H2O. Spin 12K x g , 2 min. Remove 180 L of supernatant to new tube and add 20 L 3 M Na-acetate (or K-acetate). Mix and add 500 L EtOH, then incubate -20C for _ 10 min. Pellet nucleic acids by centrifugation (12K x g, 5 mn). This step will remove chlorophyll as well as any debris carried over from step 4.6) Wash pellet w/ 75% EtOH, dry and resuspend in 10-50 L TE (pH 7.5).7) Quantitate nucleic acids and assess purity (A260/A230; A260/A280). This microprep yields nucleic acids with A260/A230 2.0 and A260/A280 2.0. Although most of the nucleic acid is probably RNA, in practice there appears to be a sufficient amount of DNA for reproducible AFLP results if one follows the protocols below.1.2 Restriction of DNA(Recipe is for one sample)0.5 g genomic DNA (total nucleic acid from mini(micro)prep)5 U EcoRI5 U MseI8.0 L 5x-Pharmacia One-Phor-All+ buffer 10x = 100 mM Tris-acetate, pH 7.5, 100 mM Mg-acetate, 500 mM K-acetate) w/ 250 ng/g BSA (=5x OPA+- BSA)Q.S. to 40 L w/ dH20- incubate 37 C for 3 hrsNote: The digestion should not be done for significantly longer than 3 hrs, as it is necessary to have active enzymes present during the ligation step (Sec. 1.3) to ensure complete digestion and ligation1.3 Ligation of adaptersAdapter PreparationEcoRI-adapter = 5-CTCGTAGACTGCGTACC (EcoRI-oligo.1) CTGACGCATGGTTAA-5 (EcoRI-oligo.2)- mix 1.7 g EcoRI-oligo.1 and 1.5 g EcoRI-oligo.2, 3 l 10X One-Phor-All+,Q.S. to 60 L w/ dH2O, heat to 95 C, and allow to cool to RT slowly. This gives a final concentration of 5 pmoles/L and makes enough adapter for 60 ligations (see below).MseI-adapter = 5-GACGATGAGTCCTGAG (MseI-oligo.1) TACTCAGGACTCAT-5 (MseI-oligo.2)- mix 16 g MseI-oligo.1 and 14 g MseI-oligo.2, 3 l 10X One-Phor-All+,Q.S. to 60 L w/ dH2O, heat to 95 C, and allow to cool to RT slowly. This gives a final concentration of 50 pmoles/L and makes enough adapter for 60 ligations.Note: Adapter oligos should not be phosphylated, this prevents adater self ligation. Both adapters are engineered such that the ligation kills the restriction site to which the adapter is ligated.Ligation RxnAdd 10 L of following mix to each 40 L digestion rxn:(recipe is for one ligation rxn)1.0 L EcoRI-adapter1.0 L MseI-adapter1.0 L 10 mM ATP4.0 L 5x-OPA+-BSA1 U T4 ligase (Pharmacia)Q.S. to 10 L w/ dH20- Incubate 37 C for 3 hrs to O/N2.0 Pre-amplification of template DNANote 1: This pre-amplification step helps to clean up some background noise that is observed on autorads of gels where non-preamplified DNA was used as a template. Furthermore, for every 1 L of total ligated nucleic acid that is preamplified one will have 150 L of template for further amplification (see below).Note 2: For this and subsequent PCR we use an MJ Research thermocycler (# PTC100; Watertown, MA, 1-800-729-2165). This thermocycler accepts 96-well microtiter plates, which is a more efficient way to amplify the multiple samples required for AFLP.Note 3: The AFLP procedure reported in NAR by Vos et al. (1995) uses a preamplification with both EcoRI and MseI primers having 1-bp, 3-extensions, followed by AFLP-PCR with primers having 3-bp, 3-extensions, whereas our preamplification uses only our EcoRI-oligo.1 (adapter oligo with no extension) (see below), and our AFLP-PCR uses only 2-bp, 3-extensions (see 2.2). Life Technology and Perkin Elmer protocols use the Vos et al. (1995) preamplification and AFLP-PCR. In our experience the single primer amplification (EcoRI-oligo.1) works quite well in comparison to the 1-bp two primer preamplification without reducing the number of polymorphic bands in the subsequent AFLP-PCR reactions. However, it should be noted that there is alot of room for personalizing the preamplification and AFLP-PCR steps to fit ones needs.Pre-amplification cocktail(recipe is for one rxn)0.5 L EcoRI-oligo.1 ( 50 ng/L)0.8 L 5 mM dNTPs2.0 L 10x-Promega Taq buffer 500 mM KCL, 100 mM Tris-HCl, pH 9.0 ( 25 C), 1.0% Triton X-100; Mg-free)1.2 L 25 mM MgCl20.08 L (0.4 U) Taq Pol (Promega)14.42 L dH2OPCR reactions(recipe is per rxn)1 L cut/ligated nucleic acid (from above) equals 10 ng total nucleic acid)19 L pre-amplification cocktail- amplify using PRE-AFLP as listed below:PRE-AFLP thermocycle profile1) 94 C, 2 min2) 94 C, 30 seci3) 50 C, 30 sec4) 72 C, 1 min5) repeat steps 2 to 4, 34 times6) hold 4 C- Do NOT perform a hot start.-dilute PCR products 1:10 w/ TE (pH 7.5) and store 4 C (or -20 C for long term) -2.1 Primer labeling (w/ 33P or 32P)(recipe is for a 50 L labeling rxn)10.0 L EcoRI-primer ( 50 ng/L)10.0 L g33P-ATP (or g32P-ATP) 3000 Ci/mmol5.0 L 10x-OPA+ (no BSA)1.0 L (5-10 U/L) T4 polynucleotide kinase (PNK)24.0 L dH2O50 L labeling rxn makes enough hot primer for 100 PCR rxns. The basic EcoRI-primer is: 5-AGA CTG CGT ACC AAT TCx yz-3where, x, y, and z represent the selective bases on the 3 end of the oligo.We typically use only 2 bp selective extensions (x and y, no z in our primers). x, y, and z are specific and constant bases within a single primer but vary from primer to primer. Thus with 2 bp selective extensions one will have 16 independent primers.2.2 AFLP-PCRAFLP cocktail(recipe is for one rxn)0.5 L labeled EcoRI-primer ( 10 ng/L)0.6 L unlabeled MseI-primer ( 50 ng/L)0.8 L 5 mM dNTPs2.0 L 10x-Promega Taq buffer (Mg-free)1.2 L 25 mM MgCl20.08 L (0.4 U) Taq Pol (Promega)13.82 L dH20 The basic MseI-primer is:5-GAT GAG TCC TGA GTA Axy z-3 like with the EcoRI-primer, x, y, and z represent selective bases that are specific and constant within a primer but vary between primers. We typically use 2 bp selective extensions in our MseI-primers. The Zabeau group, as well as commercially available kits use 3-bp selective extensions in combination with 1- bp selective preamplification (see 2.0, Note 3) PCR reactions(recipe is for one rxn)1 L preamplified DNAi19 L PCR cocktail- amplify using AFLP as listed below:AFLP thermocycle profile1) 94 C, 2 min2) 94 C, 30 sec3) 65 C, 30 sec, -0.7 C per cycle starting next cycle4) 72 C, 1 min5) repeat steps 2 to 4, 11 times6) 94 C, 30 sec7) 56 C, 30 sec8) 72 C, 1 min9) repeat steps 6 to 8, 25 times10) 72 C, 2 min11) hold 4 C3.0 PAGE of PCR productsSample preparation1) Mix each rxn w/ an equal vol of 2x formamide buffer (98% formamide, 10 mM EDTA, pH 8.0, plus bromophenol blue & xylene cyanol for tracking dye).2) Incubate samples 90 C for 45 min this reduces the vol and thus increases the specific activity of the samples.Electrophoresis1) Load sample on gel 5.0% acrylamide/bisacrylamide (19:1), 7.5 M urea, 1x TBE and run 40-50 W (constant wattage) 40-50 V/cm, like for sequencing gels with 1x TBE as the running buffer. can use 0.5x TBE as well but 1x TBE often gives clearer banding.2) Run samples until the xylene cyanol is about 2-3 cm from the bottom of the gel and stop. When 1x TBE is used in the gel and running buffer, and the gel is electrophoresed 50 W, it takes about 3.5-4 h for the xylene cyanol to get to the indicated position.Fixing & autoradiography1) Fix gel in 10% acetic acid for 30 min.2) Dry gel onto filter paper (E&K Scientific Products, cat# 2388-4567, 46 cm x 57 cm sheets) for _ 1 hr w/ heat.3) Generate autoradiograph using Kodak BioMax film (or like product from another supplier). This film gives nice O/N exposures when using either 33P or 32P as the label.4.0 Linkage analysisAnalyze F2 (or F3) recombinants relative to the two parent ecotypes (WT1, the genetic background of the mutant; and WT2, the polymorphic wild-type to which mutants were outcrossed, see Sec. 1.0) used as the backgrounds to generate the recombinants. We typically use Columbia as the parental stocks (WT1 line) for mutagenesis and Landsberg erecta as our WT2 line. Linkage is assessed by finding polymorphic bands specific to the WT2 parent that are missing in a large sample of F2 individuals (see below for suggested population size). The reason for looking for the absence of a WT2-specific band rather than the presence of a WT1 band is the inability to discern heterozygotes from homozygotes. For example, any F2 individual having a WT1-specific band could be either linked at one chromosome or both. The absence of a WT2-specific band requires therefore that the DNA specified by that polymorphic marker is homozygous for WT1 DNA in the F2 recombinant; or both chromosomes contain WT1 DNA that is linked to the locus of interest. The pitfall of this approach is that the pre