AYeastSecretionTrapAssayforIdentificationofSecretedProteins...
Secreted proteins from plants and phytopathogens play important roles in their interactions and contribute to elaborate mechanisms of attack, defense, and counter-defense, as well as surveillance and signaling. There is therefore considerable interest in developing techniques to characterize “secretomes.” Here, we describe the use of the yeast secretion trap (YST) functional screen to isolate and identify s......閱讀全文
A－Yeast－Secretion－Trap－Assay－for－Identification－of－Secreted－Proteins－...
Secreted proteins from plants and phytopathogens play important roles in their interactions and contribute to elaborate mechanisms of attack, defe
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
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
Preserving－yeast－cultures
Short term storageYeast cultures are stable for 1-2 weeks when refrigerated. Petri dishes should be sealed or in plastic bags.Medium term storageYeast
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
yeast:Assaying－mating
SetupYou have yeast strains that are deficient in mating (eg Ste12 knockouts) and would like to test whether transforming them with a plasmid that con
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
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
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 NaOH/1%
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
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 m
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
Decontamination－of－cells－from－the－yeast
I???? Destroy yeast1.???? Aspirate medium and wash cell in PBS.2.???? Incubate cells at 37oC for 5 min in non-diluted antibiotic-antimycotic.3.???? In
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
Plasmid－isolation－from－yeast
Pick colonies into 0.5ml of SD-Leu (or other appropriate SD medium)Vortex for 1minLeave to grow O/N for 18-24h at 30°C, 230-250rpm (best in 5ml bijou)
離子阱質譜儀（Ion－trap，IT）
A：離子產物掃描法（Production scanning）是蛋白質組學研究里最常用的MS/MS質譜檢測策略。該方法的目的就是要獲得蛋白質片段離子的質譜圖，然后據此鑒定出蛋白質的氨基酸序列。在本試驗中，第一個質譜儀MS1是用來篩選出某一特定的母離子。隨后，被選出的母離子在碰撞池中經由碰撞誘導解離作用
離子阱質譜儀（Ion－trap，IT）
在離子阱質譜儀中，可以捕獲離子，因此也可以積累離子。離子阱技術具有無法比擬的高靈敏度和快速數據采集能力。將離子阱技術與數據依賴性采集技術（data-dependent acquisition）結合起來，我們就能進行高通量的質譜檢測。不過，離子阱質譜儀的分辨率有限，捕獲離子的能力不高，再加上空間電荷效
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
蛋白質相互作用
Interaction Trap/Trap Two-Hybrid System·?????????Yeast Two-Hybrid System?(Finley Lab)This is one of the most comprehensive and detailed guide to yeast
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
Sucrose－Density－Gradient－Fractionation－of－Yeast－Membranes
Grow a 2 ml culture, 24 hr. at 30oC in selective mediaWhen culture is ready, use it to inoculate about 55 ml (50 ml plus 5 for O.D.600 readings) of se
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.
Snf1－in－Yeast－Glucose－Repression/Derepression
The Snf1 protein kinase is a central component of the signalling pathway for glucose repression in yeast. On removal of glucose, gene repression is re
High－Molecular－Weight－Yeast－Liquid－DNA－Preparation
Purpose:To isolate intact, high molecular weight DNA from yeast cells for subcloning and rare cutting restriction enzyme analysis. One can expect a yi
Restriction－Digests－of－High－Molecular－Weight－Yeast－DNA
Purpose:To perform restriction digests of YACs for mapping using rare cutting enzymes or more conventional restriction endonuclease digestion.Time req
Method:－Removal－of－Yeast－Contamination－from－Lymphoblast－Cultures
Purpose:This method is advantageous for saving the occasional cultures that become contaminated. Yeast contaminated cultures will appear cloudy when s
Small－Scale－Yeast－Whole－Cell－Extract－for－IP
Grow 100 ml yeast cells in desired media overnight to an A600 of ~1.0 (0.6 to 1.2 works well).?For growth in minimal media, 1 ml of a saturated overni