实验基础

Stabilize RNAStart with 15 ml E. coli Culture containing 7.undefined 109 cells (OD600= 0.2 Dilute cells or scale up)Pipet 30 ml of RNAProtect Bacteria Reagent (Qiagen) into a 50ml polypropylene conical tube. ...

Note: This procedure does not work well with Gram + cocci. Transfer 1.5 mL overnight culture to a 1.5 mL microfuge tube centrifuge for 30 sec decant supernatant.Resuspend cells in 400 mL TE by vortexi ...

Part A. Isolation of Nucleic AcidsNOTE: CAUTION! STEPS 1-10 SHOULD BE PERFORMED USING APPROPRIATE PROCEDURES FOR HANDLING MATERIAL POTENTIALLY CONTAMINATED WITH MYCOBACTERIUM TUBERCULOSIS. ALSOTHE LID ...

3 g nodules (fresh or frozen in liquid N2) were ground to a powder in mortar and pestle with liquid N2. To the powder was added ice cold 0.5 M mannitol (or sucrose) 0.05 M Tris (pH 7.5) 0.02 M Na succ ...

DNA Preparation E. coli chromosomal DNA is prepared following the method of Heath et al. ( J. Bacteriol. 174 1992). Cells are embedded in agarose then treated with deter gents and enzymes which remove ...

Some of these notes describe what may be established routines in older laboratories but are either unpublished or published in inaccessible places or scattered through the old literature and thus may ...

Isolation of DNA from filamentous fungi for PCR analysis is usually time consuming and involves use of toxic chemicals such as phenol/chloroform. In Saccharomyces cerevisiae PCR assay can be performed ...

It is well known that multiple auxotrophic markers impede fruiting in Coprinus cinereus. Restriction fragment length polymorphisms have been used to advantage (Cassidy et al. 1984 Curr. Genet. 8:607-6 ...

OverviewHigh throughput of many fungal isolates can be achieved by growing axenic cultures in either (a) 1.5mL microfuge tubes half full with liquid media (500uL) with a hole punched through the top f ...

Three transformation systems have been reported for the rice blast fungus Magnaporthe grisea (Parsons et al. 1987 Proc. Natl. Acad. Sci. USA 84:4161-4165; Daboussi et al. 1989 Curr. Genet. 15:453-456; ...

Human-pathogenic fungi such as Coccidioides immitis and Histoplasma capsulatum must be handled in Biosafety level 3 containment facilities which make for a very awkward working environment (J.Y. Richm ...

The polymerase chain reaction (PCR) is a method for amplifying specific segments of DNA defined by the small primers used to start the reaction. Using arbitrarily chosen 10-base primers one can genera ...

We have found that the DNA extraction procedure of Metzenberg and Baitch (Neurospora Newsl. 28:20)/Stevens and Metzenberg (Neurospora Newsl. 29:27) while giving excellent yield and size of DNA is some ...

Preparation of fungi for DNA extraction typically involves growing cultures in liquid culture in Erlenmeyer flasks Roux bottles or even microfuge tubes (Cenis 1992 Nucl. Acids Res. 20:2380). Growing f ...

Most RNA isolation techniques currently in use have been developed for the processing of large quantities of material. These typically involve multiple phenol extractions (Reinert et al. 1981 Mol. Cel ...

We have developed a method for isolating high quality total RNA from N. crassa mycelia that reliably yields large quantities. It is possible to extract more than 50 minipreps at once. Procedures of RN ...

Molecular biology experiments often require preparation of small amounts of DNA from many samples. This abbreviated DNA isolation method yields an average of 0.6 micrograms of genomic DNA that is suit ...

Most methods of DNA preparation from fungi are time-consuming due to the need to first make protoplasts expensive for chemicals such as cesium chloride or suitable only for small scale preparations. W ...

-Set up 60 mm dishes of P11 cells to be 100 confluent at time of infection.-Remove medium from dishes add 0.2 to 0.5 ml virus and adsorb for 30 � 60 minutes at room temperature rocking dishes occasio ...

adapted from Cell Biology A Lab Manual second edition volume 1-Grow up P11 cells in 15 cm plates to 70 � 80% confluence.-Infect cells with a MOI (multiplicity of infection) of 1 � 10 PFU per cell (o ...