LargeScaleTubulinPreparation——2
III. Pouring a 1L Phosphocellulose (PC) ColumnResin: Whatman P11 Cellulose Phosphate -- fibrous cation exchanger(1 gram of PC swells to about 4 ml packed resin)Summary: To pour a 1L column, start with 220 grams dry resin divided into 5 aliquots of 44 grams. Treat each aliquot with acid/base in a 2L beaker as described below. Older procedures described the use of large Buchner funnels to rapidly remove the acid/base. ......阅读全文
Large－Scale－Tubulin－Preparation
Tubulin is purified from bovine/porcine brain by two cycles of polymerization/depolymerization followed by removal of copurifying proteins on a phosph
Large－Scale－Tubulin－Preparation——2
III. Pouring a 1L Phosphocellulose (PC) ColumnResin: Whatman P11 Cellulose Phosphate -- fibrous cation exchanger(1 gram of PC swells to about 4 ml pac
Largescale－Immunocytology
This protocol describes our method for preparing cells for immunofluorescence, in which all incubations and washes are performed in microtiter dishes.
Preparation－of－tubulin
Although many protocols for tubulin preparation are available, the procedure described below is the simplest and highest yielding preparation I have d
Large－Scale－Plasmid－Preps:－PEG－method
1. Grow 250 500 mL of bacteria overnight in LB with 50 µg/mL of ampicillin.2. Transfer culture to Nalgene bottles. Spin in SGA rotor at 6000 rpm, at
Preparation－of－tubulin2
DAY 2: Cycling preparation of MT protein.Keep the brains in an evacuated plastic ziplock bag buried in ice from the time of slaughter during transport
Large－Scale－Plasmid－Preps:－Qiagen/Cesium－Method
Most plasmids can be adequately prepped by kits containing DNA binding columns. These columns do not do a great job of separating plasmid DNA from con
Large－Scale－Plasmid,－Cosmid,－BAC,－PAC,－and－Fosmid－DNA－Isolation
DNA Isolation by a Cleared Lysate Method Followed by Double Acetate Precipitation Version 3b - updated September 26, 1999The Most Recent Roe Lab Imple
BAC－DNA分离方法－Isolation－of－BAC－DNA－from－Largescale－Cultures
Isolation of BAC DNA from Large-scale CulturesJoseph SambrookPeter Maccallum Cancer Institute and The University of Melbourne, AustraliaDavid W. Russe
Tubulin－Basics
I. Useful Values1 mg/ml tubulin = 10 µM (assuming MW of ab-tubulin heterodimer is 100,000; in reality it is ~110,000 but almost all tubulin labs use t
Tubulin－Basics
I. Useful Values1 mg/ml tubulin = 10 µM (assuming MW of ab-tubulin heterodimer is 100,000; in reality it is ~110,000 but almost all tubulin labs use t
M13噬菌体
· M13 Phage (Michael Blaber)Very useful background information about M13: its infection, replication, packing, cloning. If you are new to phag
质粒的大量制备
· Plasmid Mini and Maxi Prep Methods (Gimila Lab) · Maxi-preps and all media, solutions (NWFSC)Isolation of cosmid, plasmid and P1 DNA
质粒的大量制备
· Plasmid Mini and Maxi Prep Methods (Gimila Lab) · Maxi-preps and all media, solutions (NWFSC)Isolation of cosmid, plasmid and P1 DNA
噬粒（phagemid）
Phagemid Related Protocol (Erik)provides procedures for phagemid lysate preparation, titering phagemid, determining uracil incorperation and SS DNA pr
Tubulin－Preparat
Materials3 - 5 Fresh Pig Brains1 M GTP1 M Magnesium SulfatePM buffer =100 mM Pipes, pH 6.9 2 mM EGTA 1 mM Magnesium Sulfate2 mM DTTPM-4M Buffer =100 m
Recycling－Tubulin
Recycling TubulinWe "recycle" tubulin fractions stored at -80¡C after the PC column and store the recycled tubulin in small aliquots for day-to-day us
酵母转化
· Yeast Transformation (Gietz Lab)LiAc/SS-DNA/PEG Transformation· Yeast Transformation (Breeden Lab)LiAc method· Large-Scale Y
Microtubule－(MT)/Organelle－Motility－Assays
Rapidly thaw and immediately place on ice one aliquot each of axonemes, Golgi or ER membranes, 45 uM tubulin, rat liver cytosol, and 20x energy regene
细菌人工染色体
The Construction of Bacterial Artificial Chromosome (BAC) Libraries (complete manuscript) (Clemson University Genomics Institute) Construction of BAC
DNA测序
DNA测序（主要内容如下）· Sequencing Gel Preparation· Preparation of Templates · DNA Sequencing by the Dideoxy Method· DNA Sequen
Immunofluorescent－Localization－of－Tubulin
LEVEL IIMaterialsCoverslip cultures of an appropriate monolayer cell linePhosphate buffered saline (PBS)Acetone/Methanol (absolute) in a 50:50 volume
酵母人工染色体
· Easy YAC Preparation Method (Andrew Davies,Shaw lab)· Screening YAC libraries (Donis Keller Lab)This is a method for screening YAC l
Labeling－Tubulin－and－Quantifying－Labeling－Stoichiometry2
II. Labeling ProtocolThe procedure described below can be scaled down if desired. It is essential to perform all steps involving caged dyes under a sa
粘粒
Preparation of Cosmid DNAPreparation of Cosmid DNA from 50 ml Cultures (Donis Keller Lab)Cosmid vectors containing foreign DNA inserts are known to re
粘粒
Preparation of Cosmid DNAPreparation of Cosmid DNA from 50 ml Cultures (Donis Keller Lab)Cosmid vectors containing foreign DNA inserts are known to re
porcine－brain－tubulin－prep
Although many protocols for tubulin preparation are available, the procedure described below is the simplest and highest yielding preparation I have d
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 (
PCR的下游应用
· Agarose Gel Electrophoresis of PCR Products (Robert H. Cruickshank)· Agarose Gel Electrophoresis of PCR Products (Immunology Resourc
Construction－and－Manipulation－of－LargeInsert－Bacterial－Clone－Libraries
Acknowledgements The organizer of the workshop acknowledges Dr. Murray Milford, Professor and Interim Head, and Dr. Mark Hussey, Professor and Interim