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TA Cloning exploits the terminal transferase activity of some DNA polymerases such as Taq polymerase. This enzyme adds a single, 3'-A overhang to each end of the PCR product. This makes it possible to clone this PCR product directly into a linearized cloning vector with single, 3'-T overhangs. The PCR products with dA overhang, are mixed with this vector in high proportion. The complementary overhangs of "T" vector and PCR product will be ligated under the action of T4 DNA ligase.

UV shadowing is a technique for visualizing nucleic acids separated on acrylamide/urea gels. The technique utilizes shortwave UV light (254 nm) and a fluor-coated TLC plate. We recommend UV shadowing as the method of choice for gel purification of nonisotopic probes synthesized with Ambion's MAXIscript™ and BrightStar™ Psoralen-Biotin Kits. The alternative to UV shadowing is staining with ethidium bromide or acridine orange and requires subsequent extraction of the dye. The detection limit of UV

The pGL3 Luciferase Reporter Vectors provide a basis for the quantitative analysis of factors that potentially regulate mammalian gene expression. These factors may be cis-acting, such as promoters and enhancers, or trans-acting, such as various DNA-binding factors. The backbone of t ...

Salmon Sperm DNA1.Dissolve 1 g salmon sperm DNA in 100 ml H2 O. 2.Autoclave (20 minutes) and aliquot in 1 ml/tube 3.Store at -20℃. (common freezer for stock solutions)

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 ...

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 ...

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Protocol for Annealing OligonucleotidesOligo Name:Lot Number:Total nmol:Volume of Annealing Buffer added:Oligo Name:Lot Number:Total nmol:Volume of Annealing Buffer added:Annealing Buffer: 10mM Tris, pH 7.5 - 8.0, 50mM NaCl, 1mM EDTA1xTE Buffer: 10mM Tris, pH 7.5 - 8.0,1mM EDTA.1.R ...

FINGERPRINTING PROTOCOL (AGAROSE GEL) * 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 ...

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ECK Description This method was suggested in a BRL "Focus" article several years ago (Zeugin & Hartley, 1985). It is a useful way to avoid coprecipitation of proteins and accumulation of salt in the final dried preparation. Protocol 1. Crude preparations: Add 0.5 volumes of 7.5 M NH4OAc. Pure preparations: Add 0.1 volumes of the same. 2. Add an amount of 95% ethanol equal to 2.5 times the new volume. 3. Continue per your favorite protocol. Note that a subsequent BRL article (-, 1982) pointed

An end labeled DNA probe is incubated with a purified DNA-binding factor or with a protein extract. The unprotected DNA is then digested with DNase I such that on average every DNA molecule is c ...

Bst DNA polymerase-catalyzed radiolabeled two-step sequencing reactions (26) are modified from those presented earlier (25) by altering the absolute amounts and the relative deoxy/dideoxynucleotide ra ...

'Phenol' as used is Analar grade. Phenol should be melted at 65℃,8-hydroxyquinoline added to a final concentration of 0.1%, and equilibrated three times with an equal volume of 1M Tris.HCl, pH 7.0. The final Tris wash is replaced with TE (10mM Tris, 1mM EDTA, pH8.0) and the phenol stored in the dark at 4℃. 8-hydroxyquinoline is added to prevent oxidation of the phenol and to act as an inhibitor of RNases. 'Chloroform' as used is a water saturated 24:1(v/v) mixture of chloroform and isoamyl alco

Protocol For the original protocol describing CGH see the article by Kallioniemi et al . For an example of how this technique has been utilized by the NCI Prostate Cancer Working Group see the section on Cell Lines.DNA RNAProteomicDNA sequence copy number changes throughout the genome in a single ...

PLATE 溶液： 40 ％聚乙二醇（ PEG ，分子量 3350 ； Sigma P 3640 ） 0.1mol/L 醋酸锂

Wear gloves throughout and work in radiation area. Monitor area before and after use. Mix the following in an eppendorf tube: 1. 0.5 microgram oligonucleotide dissolved in H2O. 2. 3 microliters 10x kinase buffer. 3. 2 microliters 32 P ATP from ICN (>5000 ci/mmole). 4. H2O so that the final volume is 30 microliters. Add 25 units T4 polynucleotide kinase and incubate 60 min at 37 ℃. Purify labeled Oligonucleotide away from unincorporated ATP Currently, we use mini Quick Spin Oligo Columns (#1 8