实验动物技术

Efficient Transformation of Yeast - LiAc MethodDifferent strains transform at different rates with different methods. Some strains transform much better using the electroporation method. Other ones ar ...

Note: Please citeAgatep R. R.D. Kirkpatrick D.L. Parchaliuk R.A. Woods and R.D. Gietz (1998) Transformation of Saccharomyces cerevisiae by the lithium acetate/single-stranded carrier DNA/polyethylene ...

Yeast Transformationinoculate 5-10 ml culture with single colony and grow ON measure OD600nm and dilute to OD600nm = 0.3 in 50 ml grow for 2-4 hrs spin wash cells with 25 ml H2O spin resuspend in 2 ml ...

Electro-Transformation of YeastDavid AmbergGrow cells to 1X10E8 or OD600 of 1.2-1.3. Spin cells at 5000rpm for 5 min and wash pellets in an equal volume of ice cold water. Wash in 1/2 volume cold wate ...

Yeast Transformation Using Frozen Competent Cells and Single-stranded DNA as a CarrierKatherine KolorReferences: Schiestl and Gietz (1989) Curr. Genetics 16:339-346 Gietz et al (1995) Yeast 11:355-360 ...

Yeast Genomic DNA IsolationDavid AmbergGrow 100 ml culture to about 1x10 to the 8th Spin 4 x 15 mls of culture down about 3k x 3 min. Resuspend entire pool in about 6 mls ddH2O aliqoute into 1.5ml mic ...

Yeast transformation using lithium acetate (rapid method)Steve HahnLast Modified December 1997This method works well when transforming with a plasmid and is rapid. (for highest efficiency transformati ...

Rhodamine-Phalloidin/Calcofluor StainingDavid AmbergGrow 50 mls of yeast to 5x10E6. Add formadehyde to the media to 4% (33 mls. of 10%). Fix in media at temperature for 10 min. Spin down cells 2-3 K f ...

Yeast Colony PCRAkada et al. Biotechniques 28:668-674 (April 2000)MATERIALS0.25% SDS10X Colony PCR Buffer: 0.125 M Tris-HCl pH 8.50.5625 M KCl25 mM MgCl210 mM dNTP's20% Triton X-100Taq polymeraseTwo G ...

Gene Disruption via PCR 14:115-132 (1998).Reaction mix:5 µl 10X Taq Buffer5 µl 25 mM MgCl22 µl 10 mM dNTP's10-100 ng template DNA25 pmols of each primer0.5 µl Taq polymerase (2 Units)_________________ ...

Quick Yeast DNA Prep: Isolation of Total DNA (genomic and plasmid) Linda Hoskins Hahn Lab 10/16/98Grow a 5 ml YPD O/N culture inoculated with a single yeast colony at 30 deg. Transfer culture to a sma ...

Genomic DNA Miniprepgrow 5 ml cells over night at 30 °C spin wash once with 1 ml H2O resuspend in 500 µl lysis buffer add acid washed glass beads to 1.25 ml vortex 2 min recover liquid phase with blue ...

Yeast Genomic DNA Prep Linda Hoskins/Hahn lab Aug 18 1997(modified from Philippsen 1991)Grow 10ml YPD cultures o/n. Figure out cell density; inoculate 30 ml YPD and grow o/n so that cell density is ap ...

Pick a medium size yeast colony (~3 mm dia) and transfer to 200 ul of lysis buffer. Add an equal volume of glass beads (0.45 mm dia). Mix on vortex at top speed for 1 min. Add 200 ul of phenol/CHCl3 ...

Yeast Miniprepfrom Robzyk and Kassir Nucleic Acid Res. (1992) 20:3790Materials:• 10 ml overnight culture yeast in drop out media (must be very dense)• STET (8% sucrose 50 mM Tris pH 8 50 mM EDTA 5% Tr ...

High Efficency Yeast Plasmid Rescue(by Trey Powers Fields Lab) Grow yeast overnight.Spin down 1.5 ml of culture in microfuge tubes.DecantAdd 250 µl of Qiagen buffer P1 to each tubeAdd about 250 µl of ...

Yeast Prep for FACS 7(13):4335-4346; 1988.1. Spin down 1E7 cells in a microfuge tube for 1 minute.2. Resuspend pellet in 1 ml of 70% EtOH. Fix for at least 60 minutes at room temperature (or up to sev ...

Yeast Cell LysatesModified from the Rine LabGrow cells to 0.3 to 0.5 O.D.(600) (~1E7 cells/ml). Use about 1.0 to 5.0 OD of cells per lysate; if your cells are at 0.5 OD per ml use 2 ml to give you 1.0 ...

Preparation of Yeast Protein Extracts ...

Yeast Protein Isolation "Yaffe-Schatz"David AmbergAdapted from Yaffe M.P. and Schatz G. PNAS 1984 81 4819-4823.Grow 25mls yeast cells to 5x10E6. Sequentially spin down cells in a 15 ml polypro tube. W ...