实验动物技术

Yeast Nuclear Extract (Small Scale) Hahn Lab (adapted from J. Leatherwood Ptashne lab)last modified Mon Aug 27 2001Cell Growth3 liters of cells are grown in YPD (3% glucose) to A600 of 3-5. Antifoam ...

Yeast RNA Miniprepgrow cells to mid-log phase (10-20 ml) (or take samples from time courses 5-25 ml) spin cells discard supernatant wash cells once with 1 ml cold RNA buffer freeze pellet in dry ice ( ...

Pol III whole cell yeast extract (small scale)Mike Schultz Pavel Aprikian Steve Hahnlast modified6/28/99Grow 2 liters yeast in YPD (with 3% glucose) to an A600 of 1.5 to 2.0 (inoculate ~1.5 ml satura ...

Curing strains of endogenous 2-micron plasmidIn order to create a circle-zero derivative from a Cir+ strain the method of Rose and Broach (1990) is used. A 2-micron-based plasmid YEp351-GAL-FLP1 * tha ...

Rapid Yeast Protein Prep for SDS PAGE and WesternHorvath and Riezman Yeast 1994; Gottschling LabSample Buffer: 10 ml0.06M Tris-HCl pH 6.80.6 ml 1M Tris 6.810% (v/v) glycerol2 ml 50% glycerol2% (w/v ...

EMS Mutagenesis of Yeast Linda Hoskins Hahn Lab Last modified10/16/981. Grow a 50 ml O/N YPD yeast culture at 30 degrees C.2. Determine cell density and transfer an amount of culture corresponding to ...

ß - GAL Filter Assaystreak yeasts to single colony replica plate onto NC-filter (leave 10 sec.) use tweezers to lift filter and slowly submerge in liquid Nitrogen for 1 min place on back side of a pet ...

Making library DNA from the DNA we send youThe original library was distributed as 18 individual pools. After 8/98 there is a new library that is distributed as 10 pools. You will be sent about a mi ...

mTn-4xHA/lacZ TRTn3 terminal inverted repeatsHAHemagglutinin (HA) epitope loxRlox site target for Cre recombinaselacZ5'-truncated lacZ gene encoding beta-galactosidaseURA3URA3 gene from S. cerevisiaet ...

Mutagenizing a yeast gene with a mini-transposonFirst clone gene into vector pHSS6. Then:Day 1: Transform plasmid into bacteria expressing transposase. Day 2: Mate transformants to bacteria carrying m ...

Making library DNA from the DNA we send youThe library is distributed as individual pools in the form of DNA. You will be sent about a microgram of each pool available. Transform a suitable amount in ...

mTn-3xHA/lacZ TR Tn3 terminal inverted repeatsXaFactor Xa cleavage recognition siteloxRlox site target for Cre recombinaselacZ5'-truncated lacZ gene encoding beta-galactosidaseURA3URA3 gene from S. ce ...

mTn-lacZ/LEU2 TRTn3 terminal inverted repeatslacZ5'-truncated lacZ gene encoding beta-galactosidaseLEU2LEU2 gene from S. cerevisiaeampEncodes beta-lactamaseloxPlox site target for Cre recombinaseUses: ...

Quantitative Mating AssayGuthrie and Fink Guide to Yeast Genetics and Molecular Biology 19911. Grow a culture of the strain whose mating efficiency is to be determined to a density of about 1E7 cells/ ...

Replication timing by comparative hybridizationM. K. RaghuramanReference: Friedman K. L. M. K. Raghuraman W. L. Fangman and B. J. Brewer (1995) Analysis of the temporal program of replication initiati ...

Replication timing by density transfer M. K. Raghuraman 1. Grow cells at least 7 generations in0.1% (w/v) C-acetate or glucose 0.01% (w/v) N-ammonium sulfate Amino acids/supplements (as required added ...

UV Mutagenesis Materials:• Cells containing ADE3-URA3 plasmid grown in URA • dropout media • YEPD plates• sterile water• petri dishes• UV light source• “light-proof” boxProcedure:1) Grow cells 2-3 day ...

Replication timing using transient hemimethylationKatherine FriedmanReference: Friedman K. L. M. K. Raghuraman W. L. Fangman and B. J. Brewer (1995) Analysis of the temporal program of replication ini ...

Yeast Cell Cycle by Flow CytometrySusan Forsburg's method for Schizosaccharomyces pombe ReagentsCold absolute ethanol. 0.5 M Na citrate stock (filtered) 50mM diluted stock. 10 mg/ml RNase A (Boil 10 m ...

Actin Capture AssayDavid AmbergDialyze purified GST fusion proteins and actin into PBS + 1mM MgCl2 . Mix 5ug actin into 50ul total volume binding buffer. Mix 5ug GST-fusion protein into total volume 5 ...