Alternative methods of preparing whole-cell DNA from fungi for dot-blot, restriction analysis, and colony filter hybridization.
There is a large and increasing number of methods for preparing whole-cell DNA from fungi. Modifications have evolved for two reasons. This first is to simplify the protocol as much as possible to allow processing of large sample numbers, in some cases for very specific uses, e.g., dot-blots. The second is to increase the quality of the DNA. Most preparations are contaminated with varying amounts of polysaccharides and unknown wall contaminants that can inhibit subsequent restriction or ligation. The extent of contamination varies with the species, the individual isolate, and at least in Neurospora, with the method or extent of growth. This paper offers three new methods. The first is a simplified procedure for isolating denatured DNAs from filamentous fungi for dot-blot analysis. The second is a rapid method for isolating DNAs from large numbers of small- to medium-scale cultures of filamentous fungi. These preparations are sufficiently pure for a variety of enzymatic reactions. The third is a nonenzymatic method for yeast colony filter hybridization that is simple, inexpensive, and efficient and results in uniform signals for a variety of species.
Min
J
J
Department of Biochemistry, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Arganoza
M T
MT
Ohrnberger
J
J
Xu
C
C
Akins
R A
RA
D001230:Aspergillus / Q000235:genetics / Q000378:metabolism; D015139:Blotting, Southern / Q000379:methods; D002175:Candida / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D004271:DNA, Fungal / Q000096:biosynthesis* / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel / Q000379:methods; D004926:Escherichia coli; D005612:Freeze Drying / Q000379:methods; D005658:Fungi / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007202:Indicators and Reagents; D009491:Neurospora / Q000235:genetics / Q000378:metabolism; D015183:Restriction Mapping; D013104:Spheroplasts
D004271:DNA, Fungal; D007202:Indicators and Reagents