LargeScalePlasmid,Cosmid,BAC,PAC,andFosmidDNAIsolation
DNA Isolation by a Cleared Lysate Method Followed by Double Acetate Precipitation Version 3b - updated September 26, 1999The Most Recent Roe Lab Implementation (by Feng Chen, Hau-Qin Pan and Fu Ying) Adapted from the Genome Sequencing Center, Washington Univ. ProtocolA smear of colonies of cosmid/BAC/PAC were picked and transferred into a 12X75 mm Falcon tube containing 3 ml of LB medium (10 g Bacto-Tryptone, 5 g Bac......阅读全文
Using－GenBank
GenBank(R) is a comprehensive database of publicly available DNA sequences for more than 205,000 named organisms and for more than 60,000 within t
A－protocol－for－cleaning－and－reusing－the－large－25－x－25－cm－plates
We regularly reuse our large 25x25 cm plating trays; initially, however, we were plagued by gross microbiological contamination when reusing the trays
Isolation－of－rodent－pancreatic－β－cells
1. Adult rats weighing 250-350g were anesthetized, sacrificed and immediately used for pancreas sampling.2. Rat islets were isolated from male wistar
Isolation－of－lymphatic－endothelial－cells
Dermal Cell Suspensions 1. Dermatomed 0.8-mm split-thickness skin was obtained from adult healthy individuals undergoing elective surgery. 2
Isolation－of－liver－infiltrating－lymphocytes
1. Hepatic T cells were isolated from fresh liver tissue collected into primary cell system.2. In the second method, tissue was diced using sterile bl
Keratocyte－isolation－(corneal－fibroblasts)
Corneal keratocytes (corneal fibroblasts) that this corneal layer is specialized fibroblasts residing in the stroma, representing about 85-90% of
Isolation－of－Microtubules－(Bovine－Brain)
LEVEL IIMaterialsFreshly removed bovine brain 2Wire sieve (tea strainer)Microtubule buffer (MT buffer)0.1 M MES (2-(N-Morphilino)ethanesulfonic acid)1
Isolation－of－Actin－and－Myosin－Filaments
LEVEL IIIMaterialsRelaxing Solution0.1 M KCl0.001 M MgCl5 mM ATP0.016 M NaHPONaHPOAdjust pH to 7.30.05 M Sodium phosphate buffer, pH 7.00.001 M EDTA (
Cajal－Body－Isolation－Protocol
Buffers and solutions(All solutions are supplemented with Complete Protease inhibitor tablet (Roche, Cat no: 1-873-580) at the final concentration of
T－and－B－cell－Isolation
Reagents Heparin - 1000 U/ml Ficoll-Hypaque PBS RPMI-1640 supplemented with 10 mM glutamine and 15% FBS AET (0.14M) Dissolve 1.967 g AET in 35 ml di-H
Lipoprotein－Isolation－－First－week
As insects have an open circulatory system, the hemolymph can be simply collected through an incision in the body wall. Most conveniently, you should
Human－FastTrack－mRNA－Isolation
Preparation of Cells1.Prepare or collect between 2x107 cells for each mRNA prep (will yield about 10-20µg of mRNA). If PBMCs from a whole blood sample
Isolation－of－lymphatic－endothelial－cells
实验概要This protocols provides a general protocol for isolation of lymphatic endothelial cells.实验步骤Dermal Cell Suspensions1. Dermatomed 0.8-mm split-thic
ISOLATION－OF－RNA－FROM－BACTEROIDS
3 g nodules (fresh or frozen in liquid N2) were ground to a powder in mortar and pestle with liquid N2. To the powder was added ice cold 0.5 M mannito
Lysosome－Isolation－in－Isotonic－Sucrose
LEVEL IMATERIALSRat liverPhysiological saline (0.85% w/v NaCl)0.25 M sucrose in 10 mM Tris-HCL, pH 7.4Brendler teflon homogenizerRefrigerated preparat
In－Vivo－Imaging－of－Far2
In vivo bio-imaging Mice were anesthetized and placed in a custom-made bed, which allowed stable and reproducible imaging of the legs. In vivo scann
293fectin™－Transfection
实验概要293fectin™ is a proprietary, cationic lipid-based formulation for transfection of DNA into eukaryotic cells. 293fectin™ is optimized for transfe
Easy－Way－to－Clone－Genes－From－a－Phage－Library
Easy Way to Clone The protocol is oriented towards a C. albicans genomic library I made in Lambda Zap II on 7/97.The overall sequence of events is: •
Hybridization－of－High－Density－Arrayed－BAC－Nylon－Filter－Blots
Protocol for hybridization of high density arrayed Bacterial Artificial Chromosome nylon filter blots with 100 PCR isolated Unigene cDNA inserts, pool
Proteome－and－Phosphoproteome－Quantification－by－DIA－on－Orbitrap－（一）
Overview1) Purpose: Data Independent Acquisition (DIA) has been applied for large-scale protein and PTM quantification on the Thermo Scientific ™
RNA－FISH－on－cultured－cells－in－interphase
IntroductionFluorescence in situ hybridization (FISH) has become a widely used method in genome and molecular genetic studies. The technique is highly
Protein－Expression－and－Purification－Protocol
Step 1: Transform appropriate DNA plasmid into BL21(DE3) E. coli cells. These cells must be competent. (Protocol for how to make competent cells.)a) T
Miniprep/Kitfree－highthroughput－protocol
BackgroundThis protocol is adapted from "Molecular Cloning: A Laboratory Manual", Second Edition, Sambrook, Fritsch, and Maniatis. It is a quick, inex
定量RTPCR－（Quantitative－RTPCR）
Application: Quantitative RT-PCR is used to quantify mRNA in both relative and absolute terms. It can be applied for the quantification of mRNA expres
In－Vivo－Imaging－of－Far3
To determine the minimum dose of Katushka plasmid needed to give detectable fluorescent intensity, we decreased the amount of pTurboFP635 to 0.5 and 1
Isolation－and－culture－of－smooth－muscle－cell
1.Male Wistar rats of about 250g body wt were anesthetized with ether and were decapitated. 2.Kidneys and thoracic aorta were removed, rinsed with
Primary－brain－cell－isolation－and－culture
1. Cerebella were removed from 7-day-old mice and passed through Nitex nylon netting (80 μm pore size) into primary cell system containing 20% (v/
Primary－cardiac－fibroblast－and－cardiomyocyte－isolation
1. Ventricles were removed under sterile conditions. 2. Ventricles were placed in cold sterile primary cell culture medium, minced into approximat
Isolation－of－human－atrial－or－ventricular－cells
1. Human tissue was derived from atrial or ventricular biopsy specimens belonging to patients undergoing heart surgery.2. Isolated myocardial tissue w
Isolation－of－Human－Pulmonary－Epithelial－Cells
Isolation of Human Pulmonary Epithelial Cells 1. Pulmonary epithelial cells from human lung tissue were isolated.2. Lungs were perfused with 10