YeastLysatesforWesterns
Cells are grown for 2-3 days as 1.5ml prep. under selection for the plasmid of interest. Spin cells down 2.6K for 5min.Resuspend in 1ml 0.25m NaOH/1% 2-mercaptoethanolIncubate 10min on ice.Add 0.16ml 50% trichloroacetic acidIncubate for 10min on ice.Pellet 14K 10min.Resuspend pellet in 1ml ice cold acetone.Pellet 14K 10min.Air dry pellet and resuspend in 200-500ul SDS SB and proceed as described below in section on w......閱讀全文
Yeast－Lysates－for－Westerns
Cells are grown for 2-3 days as 1.5ml prep. under selection for the plasmid of interest. Spin cells down 2.6K for 5min. Resuspend in 1ml 0.25m N
Yeast－Ethanol－Lysates－for－SDSPAGE－and－Western－Blotting
Procedurepick one colonyinoculate in 3 ml of the appropriate mediagrow at 30° overnightpellet the cells (5 min, 5000g)wash 1X in sterile ddH2Ore- susp
Western－雜交
Western?雜交（主要內容如下）Preparing of Protein LysatesWestern BlottingFar Western BlottingSemi Dry BlottingStripping MembranesTrouble Shooting and OthersPrepa
Embryo－Lysates－－Immunoprecipitation
Embryo lysatesTake 25 embryos and place into 1.7ml centrifuge tube.Rinse once in lysis buffer (add ~ 1ml) and remove by aspirationAdd 500 μL lysis buf
Preparation－of－Phage－Lysates
Preparation of Phage LysatesInoculate 5 ml of lambda-broth in a glass culture tube with a single colony of an appropriate host strain of?E. coli. Incu
Yeast－Media
YEPD (non-selection)-1% yeast extract-2% peptone-1.5% agar (if needed for plates)After autoclaving add glucose to 2% by adding 100 ml of 20% solution
Eccles:Protein－Lysates－from－Tissue
Cell Lysis Buffer5mL 0.1M Tris HCl pH 8 (10mM)0.44g NaCl (150mM)0.02g EDTA (1mM)0.5mL nonidet P40 (1% w/v)0.05g SDS (0.1% w/v)Make up to 50mL with MQH
酵母準備
Yeast DNA PreparationYeast Genomic Preparation? (Gottschling Lab)Rapid method for yeast genomic DNA isolation??Yeast DNA Preparation (rapid glass bead
Preserving－yeast－cultures
Short term storage Yeast cultures are stable for 1-2 weeks when refrigerated. Petri dishes should be sealed or in plastic bags. Medium term stora
yeast:Assaying－mating
Setup You have yeast strains that are deficient in mating (eg Ste12 knockouts) and would like to test whether transforming them with a plasmid that
Modified－Yeast－Transformation
Inoculate cells from an overnight culture into 50 ml YEPD and incubate at 30°C with shaking. Typically, add 0.1 to 0.2 ml saturated culture in the eve
Yeast－DNA－Prep
Protocolgrow up yeast culture to appropriate density (near saturation)spin 1.5 mls of culture for 1 min in microfuge and aspirate off supernatantresus
Dropout－plates－for－yeast
Materials(Solutions are all available from the media room)200ml bottle of 2x SD200ml bottle of 4% agar -- make sure to sign it out40% glucoseCSM minus
Fast－Yeast－Transformation
Protocol: Fast yeast transformationAdd 50 μl carrier DNA to a 1.5 ml tube.scrap cells from plate and add to the carrier DNA.Add in the following order
Yeast－Nuclei－Isolation
This method gives yeast nuclei which look nearly purified microscopically. Nuclei isolated in this way do not give active transcription extracts when
Eccles:Protein－Lysates－from－Cells－in－Culture
Cell Lysis Buffer 5mL 0.1M Tris HCl pH 8 (10mM) 0.44g NaCl (150mM) 0.02g EDTA (1mM) 0.5mL nonidet P40 (1% w/v) 0.05g SDS (0.1% w/v) Make up to 50mL
Yeast－Genomic－DNA－Prep
Grow 10ml YPD cultures o/n. Figure out cell density; inoculate 30 ml YPD and grow o/n so that cell density is approximately 2 x 108cells/ml the next
Plasmid－isolation－from－yeast
Pick colonies into 0.5ml of SD-Leu (or other appropriate SD medium) Vortex for 1min Leave to grow O/N for 18-24h at 30°C, 230-250rpm (best in 5ml b
Decontamination－of－cells－from－the－yeast
I???? Destroy yeast 1.???? Aspirate medium and wash cell in PBS. 2.???? Incubate cells at 37oC for 5 min in non-diluted antibiotic-antimycotic. 3.
Endy:Yeast－Colony－PCR
MethodUsing sterile pipette tips, transfer a 1 mm colony into 50 uL of 60 U/ml Zymolyase3 uL of 1 U/mL Zymolyase stock solution47 uL of waterIncubate
Blackburn:Yeast－Colony－PCR
OverviewThis is a quick and easy yeast colony PCR protocol that does not require zymolyase step.Updated Protocol:?Blackburn Lab: Quick and Easy Yeast
Live－Cell－Imaging－of－Yeast
Live Cell Imaging of YeastDaniel R. Rines, Dominik Thomann, Jonas F. Dorn, Paul Goodwin and Peter K. SorgerINTRODUCTIONThe development of cloning vect
Yeast－Media,－Solutions－and－Stocks
Yeast Media:Note: Synthetic complete medium can be prepared by adding media supplements (see below).Medium using 6.7 g yeast nitrogen base without ami
Methods－for－the－Measurement－of－a－Bacterial－Enzyme－Activity－in－Cell－Lysates
Abstract The kinetic characteristics and regulation of aspartate carbamoyltransferase activity were studied in lysates and cell extracts of?He
Cell－and－tissue－lysis－hub
This page should point you to the many different general and lab-specific protocols describing tissue and cell lysis and serve as forum for comparison
Yeast－Cell－Cycle－by－Flow－Cytometry
ReagentsCold absolute ethanol.0.5 M Na citrate stock (filtered), 50mM diluted stock.10 mg/ml RNase A (Boil 10 mins, cool, filter and store at -20°C).4
Acid－Phenol－Yeast－RNA－Prep
This is the preferred method for yeast RNA preparationuse Gloves and RNAse free solutions throughout.1. Use a YPD overnight culture to innoculate fres
定量westerns：最佳的蛋白印跡標準化方法是什么？
現代數字檢測儀器對于western blotting不僅僅是檢測“有或沒有”的技術，還可以進行western blotting重復性和定量的檢測。 在檢測方法的線性動態范圍，對數據進行標準化以控制蛋白上樣量和膜轉印帶來的變化，可以獲得真正的定量結果。 但是，對蛋白印跡進行標準化的最佳方法是什么？
Yeast－Gene－knockout－using－Oligo/PCR
Universal primers for gene knock-out using dominant drug markers: Kan, Clonat, and Hygromisin-B.?Forward primer: 5’ TCAGGGGCATGATGTGACT 3’Reverse prim
Preparation－of－Yeast－DNA－Embedded－in－Agarose－Plugs
Preparation of Yeast DNA Embedded in Agarose PlugsAnja van Brabant(adapted from?Iadonato, S. P., and A. Gnirke. 1996. RARE-cleavage analysis of YACs.