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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 ...

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 ...

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 ...

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 ...

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. 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 ...

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）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 ...

PURPOSE: TolocateaparticularsequenceofDNAwithinacomplexmixture(locateonegenewithinanentiregenome) SeparatemixtureofDNAsequencesonagelprobewithspecificDNAsequence(gene) Otherrelatedblottingtechniques: ...

1. Add an equal volume (equal to sample volume) of P/C to sample.2. Mix (shake don't vortex).3. Take aqueous (upper) layer. (If dirty sample repeat Ph/Chl step until interface is fairly clean).4. Add ...

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 ...

IntroductionMethods and reagents is a unique monthly column that highlights current discussions in the newsgroup bionet.molbio.methds-reagnts available on the internet. A commonly occurring theme on t ...

This is a scaled up version of the Baron protocol which has been modified to achieve purity comparable to CsCl preps. I have not sorted out strain variations yet but HB101 works well.1、SolutionsSoluti ...

There are many methods for DNA preparation without using kits.Below are a few minipreps.These protocols may be scale up to midi or maxi prep if necessary.1.Ammonium Acetate Method 1)Spin down cell and ...

1.Prepare or obtain Buffered phenolpH 8.0.Add 2 crystals of 8-Hydroxyquinoline to prevent oxidation.This also identifies the organic phase as yellow-colored.2.Combine DNA sample with an equal volume o ...

1、Put a 0.5-1.5 CM MOUSE TAIL SNIP in a 1.5 ml eppindorf tube and store frozen until digestion.Make certain that the eppindorf tubes seal wellotherwise your samples may leak out during the shaking ste ...

1、Grow an overnight culture from a single colony in 2 mls LB antibiotic.2、Transfer 1.5 mls of culture to eppendorf tube.Spin for 1 minute on high and remove supernatant.3、Add 700 µl STET. Add 25 µl ...