DNA提取与纯化

Purpose: Purification of oligonucleotides (which have already been purified by reverse phase) can increase the efficiency of site directed mutagenesis from around 30% to ~75% or greater in some cases ...

PrincipleThe nucleic acids contained within the gel slice are electrophoresed out of the gel funneled down into the v-shaped channel and trapped within a high salt cushion resting at the bottom of the ...

Sample Preparation Cheek cells are obtained by rinsing the mouth with 25mls of any commercial mouth wash solution available for about 30sec the first thing in the morning. It is important not to brush ...

Materials:Silica Suspension:add 2 g of silica to 15 ml of H2Owash 3x by centifugation at 2000 x g for 2 minestimate vol of silica and resuspend in 2 vol H2OSilica Wash Solution:50 mM NaCl 10 mM Tris 7 ...

- make a 5µm section to do an evaluation of the % tumour cells- make 50X50µm sections for the DNA isolation- put sections in a falcon tube containing 10 ml of 1XSE- add 100µg/ml prot. K (endconcentrat ...

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 ...