DNA提取与纯化

1. Add 400 μl of TE buffer then 400 μl of 1-butanol to the oligonucleotide glass vial. 2. Vortex well, then spin down in tabletop centrifuge for 10 minutes at ~2,000. rpm¹s. 3. Remove top, butanol layer with a sterile pipette tip and discard. 4. Add another 400 μl of 1-butanol. 5. Vortex well, then spin down as above in tabletop centrifuge. 6. Remove top, butanol layer and discard. 7. Transfer aqueous phase into a new 1.5 ml tube. 8. Dry in a speed vac for about 5 minutes to remove all of the b

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Solutions Gel Stocks Diluent 5X Buffer 25% Acrylamide 209 g Urea 209 g Urea 209 g Urea up to 500 ml Q 250 ml 10X TBE 120.8 g Acrylamide up to 500 ml Q 4.1 g BIS up to 500 ml Q 2.5 M NH4 OAc 19.2 g NH4 OAc up to 100 ml Q Formamide Dye 9 ml deionized formamide 500 m l 10X TBE 500 m l 0.2% bromophenyl blue and 0.2% xylene cyanol Other Reagents Needed: 0.22 m M disposable syringe filters, short wave UV source, intensifying screen for UV shadowing Procedure • Pour a 20% denaturing gel: 12 ml 5X Buffe

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.

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 ...

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

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 ...

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

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 ...

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小于100bp的片断通常在电泳时就比较难观察分辨，需要用分辨率很高的琼脂糖或者丙烯酰胺凝胶。比如Cambrex的NuSieve 3:1或者GTG，或者是大名鼎鼎的MetaPhor琼脂糖。所以实际上对于小片断，可以分为两种情况：一个是真的要回收电泳中特别小的片断，这种情况我们前面有提到，比如Qiagen MinElute系列可以回收70bp以上的片断；而QIAEX纯化介质可以回收40bp以上的小片断，可以根据情况选择。 ...

DNA absorbs ultraviolet light due to its highly conjugated nature. DNA may thus be easily quantitated in a UV spectrometer. Typically 1 OD260 (i.e. a solution having an absorbance of one unit at 260 n ...

macerate tissue in Eppendorf tube without butter at RT add 400 m l extraction buffer vortex for 4 sec leave sample at RT until other samples are ready (> 1 h) spin in microfuge for 1 min transfer 300 m l of supernatant to different Eppendorf tube (prefilled with 300 m l isopropanole) mix and leave at RT for 2 min spin for 5 min vacuum dry pellet and take up in 100 m l TE use 1-2.5 m l for PCR Remarks: DNA is stable for one year at 4℃ Solutions: Extraction buffer: 200 mM Tris-HCl pH 7

The principle and procedures of RLGS method was first described by Hatada et al. (1991) and its improvement was described by Asakawa (1996). Basically based on their procedures ...

酵母 菌基因组 DNA的提取 一：仪器： 同方法一 二：试剂： SE缓冲液（1M山梨醇，0.1MEDTA pH7.5）；溶菌酶（50mg/ml）；20％PVP；蛋白酶K缓冲液（10mM Tris pH7.6 0.5% SDS 1mM EDTA）；其余同前 三：操作 1.5ml 对数生长期细菌细胞 离心，12000rpm，1-2min 沉淀 溶于590ulSE缓冲液中混匀+1 ...

PEG Preparation of Plasmid Plasmid isolated by this procedure can be used routinely for electrophoretic analysis, restriction endonuclease digestion and transformation of E. Coli., sequencing, PCR and most other molecular biological techniques. The procedure is a modification of the rapid alkaline lysis method of Ish-Horowitz and Burke (1981). You will need 3 basic solutions:- Solution I: 50mM glucose, 25mM Tris.Cl, pH 8, 10mM EDTA Solution II: 0.2M NaOH, 1%(w/v) SDS Solution III: 3M potassi