DNA实验技术

比起其它公司的原核表达系列来说Invitrogen有个非常突出的地方就是它的重组克隆技术。像之前Novagen大部分载体都是用传统的酶切、链接方式做重组质粒，但是Invitrogen的表达系统大量运用了它的TOPO技术和Gateway技术。虽然现在在许多做科研的学生看来，能出实验结果是最重要的，中间的过程不是那么重要。可是只有创新的技术才能推动科学不断的进步，比如：如果我们一直采用手工法测序，那真 ...

Electroelution of agarose fragmentsElectroelution buffer1 M Tris pH 7.5 12.0 mls0.5 M EDTA 0.24 mls1 M NaCl 3.0 mlsqs to 600 mls dH2OAcetate cushion 3 M NaAcetate pH 4.8 480 μl0.1 % Bromphenol Blue ...

Procedure 1. Grind 2 to 5 g of frozen leaves to a very fine powder with a liquid nitrogen-cooled mortar and pestle.2. Add 25 ml of CTAB Buffer and transfer to a 50 ml tube.3. Incubate at 65℃ for 20 ...

Phenol (removes protein) 1.add equal volume of Phenol (= tris-saturated Phenol-Chloroform-Isoamyethanol) 2.vortex 3.spin 2 minutes at 12000 rpm 4℃4.transfer supernatant to a fresh tube (avoid aspirati ...

Isolation of DNA from Mouse Tail Biopsies 1. Obtain tail biopsies from 2 to 3 week old mice: Hold mouse firmly at base of tail with one hand with the other cut off 0.5 to 1.0 cm of the tail tip with a ...

Protocol for Isolation of Genomic DNA from Mammalian Cells1.Trypsinize harvest and resuspend cells at 107 / ml in 10 mM Tris-HCl (pH 8.0) 10 mM EDTA. 2.Add SDS and Proteinase K to a final concentratio ...

1. Run gel (0.8 -1.0% agarose is best). For yeast chromosomal Southerns digest 20 μg DNA. Use 50 ml minigel for most purposes. Photograph gel but minimize exposure to UV light.2. Depurination: Place g ...

Below we present the protocols we have used to isolate DNA from various tissues using Silica and Guanidinium Thiocyanate.These protocols are adapted from Boom et al. (1990)Höss & Pääbo (1993)and Höss ...

Transgenic Mouse and Gene Targeting FacilityTg 008 Southern BlottingMaterials and EquipmentHybaid hybridization oven (ISC BioExpress H-9250)Vacuum oven (VWR 52201-218)Ludlum Geiger CounterRadiation Sh ...

Background on the art of getting beautiful Southerns:There are several things to consider. (1) Digesting enough DNA. (2) The DNA must be digested to completion; i.e. no partial digests because they ma ...

Day 11. Digest DNA for 6 hours (or overnight)BSA 10 mg/ml 0.5 22.65 ul of 10 ug Genomic DNARnaseA 10mg/ml 0.1 in TE mixed to 7.35 ul cocktailSpermidine 100nM 0.75 per ...

1) Take one medium sized leaf or half a large leaf (5 to 20 cm^2) weigh and freeze in liquid nitrogen.2) Grind the tissue in a bleached and baked pestle and mortar with liquid nitrogen.3) Transfer the ...

Southern Blotting: DNA Transfer1. Depurination of DNA fragments: Wash gel in 0.25 M HCl 5' (small gels) 7' (large gels)2. Denaturation: Wash gel in (0.5 M NaOH 1.5 M NaCl) for 20' (small) to 30' (larg ...

作分子生物学实验，核酸纯化、酶切、克隆算是最基本的步骤了。因为要做定向克隆，通常要作双酶切。为了省时省事儿，我们常常想方设法把两个酶放在一起切。可是事情并非总那么称心如意��两个酶经常在一起闹别扭，不是反应温度不同啦，就是Buffer不同，有时候两个酶切位点连在一起，必须先切一个再切另一个，否则就切不动��因为有的酶除了识别位点之外还需要旁边留有几个碱基，如果正好被切掉了就出问题。于是有人就到处找 ...

Add NH4Ac (10 M stock or solid) to the sample for a final concentration of 2.5 M mix (spin at 4℃ transfer the supernatant to a new tube; optional spin for extra purification of the DNA) add 2.5 volume ...

1. Separate DNA fragments in an agarose gel cast with 0.5 mg/ml Ethidium bromide. Locate bands with a hand-held long-wave UV lamp.2. Slice the gel with a razor blade above and below the bands of inter ...

Equipment and reagents1. Phenol2. TE bufferpH 8.0 (10 mM Tris-HClpH 8.0;1 mM EDTApH 8.0)3. 24:1 (v/v) chloroform-isoamyl alcohol4. 3 M potassium acetate pH 5.5prepared by adding glacial acetic acid t ...

1）基因组DNA Southern印迹的制备 预备 1．用适当的限制性内切酶消化基因组DNA样品（10μg）。 2．进行琼脂糖凝胶电泳。一般用0.7～1.0％的琼脂糖凝胶分离基因组DNA，它在1～15kb的范围内有较好的分辨率，可选用TBE或TAE缓冲液。琼脂糖凝胶电泳需在1V/cm的电压下进行，如果要分离片段大小相似的DNA带，应用较大的凝胶（20×25cm）。常用的标准品是由Hind Ⅲ消化的 ...

Author: Suzanne GerttulaDate: March 141994 Digest between 1 and 10 ug Genomic DNA. I digest at 37 C for about six hours over the course of a day. Spin down briefly heat to 65 C for 10 min and then chi ...

1. Spin down 1.5 ml of overnight culture in 2ml or 1.5ml tube for 1 minute on high. 2. Asprirate supernatant and resuspend cell pellet in 100 µl Solution I (25 mM Tris-HCl pH 8.0 10 mM EDTA). 3. Add 2 ...