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酵母遗传学技术-实验方法 |
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来源：互联网 作者：未知 发布时间：2006-12-16
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- Genome-wide Gene Expression Analysis (Richard Young Research Group,Whitehead Institute for Biomedical Research)
A genoe-wide gene expression analysis using high-density oligonucleotide arrays
- Restriction Digests of High Molecular Weight Yeast DNA (Donis Keller Lab)
Restriction digests of YACs for mapping using rare cutting enzymes or more conventional restriction endonuclease digestion.
- Preparation of Yease Cell for ImmunoEletron Microscopy (Jon Mulholland, Botstein Lab)
- Yeast Immunofluorescence with Methanol/Acetone Dehydration (Botstein Lab)
- Yeast Immunofluorescence (without methanol) (D. Amberg, Botstein Lab)
- Yeast Rhodamine-Phalloidin/Calcofluor Staining (D. Amberg, Botstein Lab)
- Yeast Cell Cycle by Flow Cytometry (Salk Institute, Flow Cytometry Laboratory)
- Vectorette PCR of Yeast DNA (Botstein Lab)
- Yeast Actin Capture Assay (D. Amberg)
- Yeast Preparation for FACS Analysis (Gottschling Lab)
- Replication Timing by Density Transfer (Fangman/Brewer lab)
- Replication Timing by Comparative Hybridization (Fangman/Brewer lab)
- Replication Timing Using Transient Hemimethylation (Fangman/Brewer lab)
- ß-GAL Filter Assay (Breeden Lab)
- Bgal filter assay (Herskowitz Lab)
- Bgal plate overlay assay (Herskowitz Lab)
This assay is semiquantitative. Cells can even be recovered from underneath the overlaid agar. The method could be used for any enzyme with a colorimetric assay.
- Bgal liquid assay (Herskowitz Lab)
- Pheromone Halo Assay (Dohlman Lab)
This is a bio-assay that measures the responsiveness of cells to a factor pheromone. The assay is easy to conduct and the results are usually unambiguous and highly reproducible.
- Liquid ß-gal Assays (Dohlman Lab)
This protocol is used to quantitatively measure the pheromone response over a range of pheromone concentrations.
- ß-gal Lift Protocol (Dohlman Lab)
This method is used to screen for and examine mutants with altered pheromone responses.
- Handling and using insertion libraries (YGAC)
- Curing strains of endogenous 2-micron
- mTn-lacZ/LEU2 insertion library
- mTn-3xHA/lacZ insertion library
- mTn-3xHA/GFP insertion library
Yeast Gene Mutagenesis
- Protocols for shuttle mutagenesis/epitope-tagging of a yeast gene with mTn-lacZ/LEU2 (Yale Yeast Analysis Center)
mTn-lacZ/leu2 can easily be inserted at mutiple sites in a given gene. The mutagenized DNA is then transformed into yeast, where it replaces the chromosomal locus by homologous recombination. The transposon insertions create a pool of insertion/disruption alleles. Insertions that generate in-frame fusion of the coding region to lacZ can be used to monitor and quantify gene expression, via assays for beta-gal activity. The fusion protein can also be immunodetected using antibodies directed against beta-gal.
- Protocols for shuttle mutagenesis/epitope-tagging of a yeast gene with mTn-3xHA/lacZ (Yale Yeast Analysis Center)
Same protocol as above
- Mutagenizing a yeast gene with an mTn (YGAC)
- Non-technical overview
- Using mTn-lacZ/LEU2
- Using mTn-3xHA/lacZ
- Using mTn-4xHA/lacZ
- Using mTn-3xHA/GFP
- UV Mutagenesis (Corbett Lab)
- EMS Mutagenesis (Herskowitz Lab)
- EMS mutagenesis of yeast (Hahn Lab)
- Generating a Temperature Sensitive Allele (Herskowitz Lab)
- Confirmation PCR (Saccharomyces Genome Deletion Project)
To study the genetics of yeast, yeast gene ORF is deleted by PCR based strategy to generate mutated strain. Confirmation PCR is used to verify if the yeast ORF deletion is successful
Introduction/General strategy
Confirmation PCR primer design
Primer organization in the 96-well plates
Oligonucleotide concentrations
Quality control procedures
Single tube PCR protocol
96-well format protocol
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