Dropoutplatesforyeast
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 the relevant marker(s), as powderStack of plates (above the -20 freezers)Yeast growth medium generally consists of three parts: a nitrogen base (supplied by SD), a sugar source (supplied by glucose) and necessary amino acids (supplied by CSM). These three components ar......阅读全文
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
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
酵母培养基和相关试剂
Yeast Hartwell's Complete (HC) Media (Gottschling Lab) Yeast Complete Media Yeast Media (PMCI) Yeast Media, Solutions and Stocks (Donis Keller
Layered－plates
General Information The cells being plated are sandwiched between layers of cell-free agar. The submerged cells grow into smaller colonies and many
Pouring－Plates
1. For rich media, weigh out appropriate ingredients and place into a flask. Add water until appropriate volume. Use a flask at least 2 times larger
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
Performing－a－hunt－by－interaction－mating
AbstractWhen more than one bait will be used to screen a single library, significant time and resources can be saved by performing the interactor hunt
Preparation－of－Agar－plates
Prepare media and add 1.5 agar before autoclaving it (15g per liter). After autoclavation, cool the media in a 55 degree waterbath. Do not allow th
Spheroplast－Transformation
MaterialsYPD platesYPD1 M sorbitol 182 g/l (Sigma S7547)2 M sorbitol 36.4 g/100 mlSCE (per liter)1 M sorbitol (182 g)100 mM citric acid trisodium salt
Preparation－of－Broth－and－Plates,－etc.
Recipes: 1) LB Broth Make 16 gm of LB Broth Base (Gibco #M27800C) up to 800 ml in ddH2O. Swirl to dissolve, then add 110 µl of 10 N NaOH. Autoc
Agar－Plates－for－Selection－of－Clones－in－Bacteria
Cloning of PCR products Stocks: LB Agar: Luria Broth after Lennox: per Liter Tryptone 10 g Yeast Extract 5 g Sodium Chlori
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－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
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
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
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
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
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
Twohybrid－analysis－of－genetic－regulatory－networks
1. Introduction and BackgroundThere is a great need for general methods to characterize the proteins that contemporary biology makes available. The li
Preparing－Antibiotics－Stock－Solution－and－Ampicillin－Agar－Plates
AMPICILLIN Beta-lactam-antibiotics are not very stable when dissolved. Slow but steady degradation happens even when frozen to -20°C. Therefore comme
酵母培养
Streaking Yeast Stocks (Donis Keller Lab)Very nice protoocol for yeast workLong-Term Storage of Yeast Stocks (Donis Keller Lab)Yeast storage and reviv
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
酵母转化的几种方法
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 m
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