DNA实验技术

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This is a very fast mini-prep protocol which is suitable for sequence analysis and restriction digests. Although the yield is higher than Protocol D.1, there is considerable chromosomal DNA, RNA and protein contamination. Solutions Sucrose/Tris 25% sucrose 25 g sucrose 50 mM Tris pH 8.0 5 ml 1M Tris pH 8.0 up to 100 ml with Q store at room temperature Triton Lysing Mix 5% Triton X-100 5 ml Triton X-100 5% sucrose 5 g sucrose 50 mM Tris pH 7.5 5 ml 1M Tris pH 7.5 50 mM EDTA 10 ml 0.5 M EDTA pH 8.

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第一节 概 述 把一个有用的目的DNA片段通过重组DNA技术，送进受体细胞中去进行繁殖和表达的工具叫载体(Vector)。细菌质粒是重组DNA技术中常用的载体。 质粒(Plasmid)是一种染色体外的稳定遗传因子，大小从1-200kb不等，为双链、闭环的DNA分子，并以超螺旋状态存在于宿主细胞中。质粒主要发现于细菌、放线菌和真菌细胞中，它具有自主复制和转录能力，能在子代细胞中保持恒定的拷贝数， ...

Materials: RPMI 1640 medium fetal calf serum (FCS) 20% Colcemid (e.g. Boehringer Mannheim cell biology reagents Best.-Nr. 295892) cell cuture flask Phythemaglutinin PHA-L (Seromed M 5030) CO2 ce ...

Restriction digests consist of: 15.75 ml ddH2 O 1 µl 10 X buffer B (Boehringer Mannheim) 0.25 µl HindIII (40 U/µl) 3 µl DNA Set up digestions in 96 well plates. Incubate at 37℃ for 4.5 hours. This can be done in a thermocycler. After digestion, a brief centrifugation will collect DNA at the bottom of wells. Seal plates with foil tape and store at 4℃ if necessary. Prepare 1% agarose gels in 1X TAE: Cool molten agarose to 46℃ in a water bath with occasional stirring. Pour into 20X25 cm UV tra

DNaseI Footprintint Solutions 10X Binding Buffer 200 mM Tris 8.0 200 m l 1M Tris pH 8.0 500 mM NaCl 100 m l 5M NaCl 10 mM EDTA 20 m l 0.5 M EDTA pH 8.0 680 m l Q store at room temperature DNaseI Dilut ...

This protocol works well from a 5 ml starting culture or a 1 ml starting culture (adjust volume accordingly). Solutions 2X YT Media 16 g tryptone 10 g yeast extract 5 g NaCl 1 ml 1N NaOH up to 1 liter with Q Ampicillin Stock (1000X) 0.15 g ampicillin 1 ml Q can be stored at 4℃ for several weeks Tetracycline Stock (1000X) 15 mg tetracycline 500 m l EtOH 500 m l Q vortex to dissolve and store at 4℃ Kanamycin Stock (1000X) 50 mg kanamycin 1 ml Q can be stored at 4℃ for several weeks 20% PEG 8000/ 2

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一、导论 已经提出过许多方法用于从细菌中提纯质粒DNA ， 这些方法都含有以下3个步骤： 细菌培养物的生长。 细菌的收获和裂解 质粒DNA 的纯化。 （一）细菌培养物的生长 从琼脂平板上挑取一个单菌落，接种到培养物中( 有含有行当抗生素的液体培养基中生长) ，然后从中纯化质粒，质粒的提纯几乎总是如此。现在使用的许多质粒载体（如pUC 系列）都能复制到很高的拷贝数，惟致只要将培养物放在标准LB 培养 ...

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Sephadex G-50 spun column purification Spin column purification can be used to change buffers without a concomitant change in solution volume to remove protein contaminants or to purify plasmid DNA s ...

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TE solution 10 mM Tris (pH to 7.5) 1 mM EDTA (pH to 8.0 to dissolve) 2.Frozen agarose gel piece containing the desired DNA fragment Supplies: 1.Micropipetter and tips 2.Microcentrifuge and tubes 3.Spatula 4.PCR machine and the 600-ul tubes for use in the machine 5.Ultrafree-MC(R) filter units, 0.45 μm Procedures: 1.Quick thaw the gel piece in a 600-μl tube using the PCR machine at 37 ℃ 2.Macerate the gel piece with a spatula. 3.Transfer into the sample cup of the filter unit. 4.Spin th

This protocol was designed to generate directionally end-labeled probes for DNaseI footprinting but it can be used for any application that requires end-labeled DNA probes. Solutions 10 mM dNTP Stocks Thaw100 mM stocks (NEB or Boehringer Mannheim) on ice and dilute 10-fold in Q. store small (10-20 ml) aliquotes at -80 degrees and thaw on ice just prior to use 10 X Klenow Buffer 0.5 M Tris 7.5 500 ml 1 M Tris 7.5 0.1 M MgCl2 100 ml 1 M MgCl2 10 mM DTT 100 ml 0.1 M DTT 0.5 mg/ml BSA 50 ml 10 mg/ml

Glass wool method: Run TAE agarose gel and cut the appropriate band out with a clean razor blade. Poke a small hole with hot needle on the bottom of an eppendorf tube, and jam the hole with a little of siliconized glass wool. Put the gel in the tube and put this tube onto another tube and spin with 5-7 Krpm for 5-10 min. Add 1/2 vol. of phenol and 1/10 vol. of 3 M NaAc of the gel volume to the agarose tube and spin for another 5 min. Discard the agarose tube. Add 1/2 vol. of chloroform to th

1. Pick single colony and inoculate 250 ml of LB broth containing 100 m g/l ampicillin or appropriate antibiotic. Shake at 250 RPM overnight.2. Centrifuge cells in a Sovall GSA (250 ml)or SLA-3000 (500 ml) rotor at 5 k × g for 10 minutes.3. Resuspend cell pellet in 5 ml of GTE buffer (50 mM Glucose, 25 mM Tris-Cl, 10 mM EDTA, pH 8) by ...

Pouring the Gel Outline: Pouring this big & thin 6% acrylamide gel ("Mother of all gels") is quite a challenge and probably the reason why smart whimps by them ready to use. Supplies & ...

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Introduction This manual is a compilation of many of the everyday methods used in the average molecular biology laboratory with emphasis on the techniques for large scale DNA sequencing protocols and ...