Nonmammalian Genomic Analysis: A Practical GuideBruce Birren, Eric Lai Offering detailed protocols for those needing to construct a variety of maps and isolate genes, this unique book is intended to popularize the new techniques of genome analysis derived from the Human Genome Project. The power of these new methods is often most striking when applied to problems outside of human genetics, particularly the nonmammalian systems on which many researchers focus. Many of these organisms are economically important and biologically rich. Nonmammalian Genomic Analysis: A Practical Guide covers the "how to" aspects of preparation, handling, cloning, and analysis of large DNA and the creation of chromosome and genome maps. This lab manual facilitates the transfer of these technologies to small "low tech" environments and allows them to be used by those with no background in genome mapping or large-fragment cloning. Like having a local expert, this collection provides procedures for anyone, anywhere, and allows the replication of others' success.
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From inside the book
Results 1-5 of 80
Page 12
... solution Tris 40 242 g Tris base Acetate 40 57.1 ml glacial acetic acid EDTA 25 100 ml 0.5 M EDTA, pH 8.0 H2O - add to bring final volume to 1 L B. Solutions 1. Bacterial lysis Solution Final concentration Stock solutions Amount needed ...
... solution Tris 40 242 g Tris base Acetate 40 57.1 ml glacial acetic acid EDTA 25 100 ml 0.5 M EDTA, pH 8.0 H2O - add to bring final volume to 1 L B. Solutions 1. Bacterial lysis Solution Final concentration Stock solutions Amount needed ...
Page 14
... solution rather than after embedding the cells in agarose. Resulting spheroplasts must be osmotically stabilized during any washing steps to prevent lysis prior to embedding in agarose. (3) Add EDTA to all solutions as early in the ...
... solution rather than after embedding the cells in agarose. Resulting spheroplasts must be osmotically stabilized during any washing steps to prevent lysis prior to embedding in agarose. (3) Add EDTA to all solutions as early in the ...
Page 15
... solution than after embedding cells in agarose. Therefore, a higher yield of chromosomes is obtained by first preparing spheroplasts in solution and then embedding these in agarose for digestion with proteinase K, as described in ...
... solution than after embedding cells in agarose. Therefore, a higher yield of chromosomes is obtained by first preparing spheroplasts in solution and then embedding these in agarose for digestion with proteinase K, as described in ...
Page 16
... solution tube. Incubate at 37°C for 2–16 hr to allow spheroplasts to form. (10) Discard the lysis solution, taking care to retain the samples, and add 4 ml digestion buffer. Incubate at 50°C for 12–36 hr with gentle agitation. (11) ...
... solution tube. Incubate at 37°C for 2–16 hr to allow spheroplasts to form. (10) Discard the lysis solution, taking care to retain the samples, and add 4 ml digestion buffer. Incubate at 50°C for 12–36 hr with gentle agitation. (11) ...
Page 17
... solution at 4°C, where they will be stable for weeks or months. Wash samples by placing in several changes of 50 mM EDTA prior to use. C. Controlling and Determining DNA Concentration The final concentration of DNA in the agarose is ...
... solution at 4°C, where they will be stable for weeks or months. Wash samples by placing in several changes of 50 mM EDTA prior to use. C. Controlling and Determining DNA Concentration The final concentration of DNA in the agarose is ...
Contents
1 | |
25 | |
61 | |
Chapter 4 Generating and Using DNA Markers in Plants | 75 |
Chapter 5 Genome Mapping of Protozoan Parasites by Linking Clones | 135 |
Chapter 6 Macrorestriction Mapping and Analysis of Bacterial Genomes | 165 |
Chapter 7 Cosmid Cloning with Small Genomes | 197 |
Chapter 8 Construction of PI Artificial Chromosome PAC Libraries from Lower Vertebrates | 223 |
Chapter 9 The Selection of ChromosomeSpecific DNA Clones from African Trypanosome Genomic Libraries | 257 |
Chapter 10 Analysis of the Dictyostelium discoideum Genome | 293 |
Chapter 11 Integrated Genome Mapping by Hybridization Techniques | 319 |
Index | 347 |
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Common terms and phrases
Acad AFLP agarose gel aliquots amplification bacterial bacteriophage bands blot buffer cells centrifuge cerevisiae chDNAs chromosomes coli colonies containing contigs cosmid Dictyostelium DNA fragments DNA molecules DNA sequence EDTA electrophoretic karyotype ethidium bromide filters Final concentration gel electrophoresis gene Genet genomic DNA hybridization identified Incubate insert isolation karyotype lane large DNA ligation linear linking clones markers method mg/ml molecular NaCl Natl Novozym Nucleic Acids Res nucleotide oligonucleotide PAC cloning partial digestion PFGE physical mapping plasmid plates plugs polymerase polymorphisms prepare primers probe procedure proteinase K protocol protoplasts pulse pulsed-field gel RAPD reaction repeat restriction digestion restriction endonuclease restriction enzyme restriction fragments restriction mapping resuspend RFLP room temperature samples selected separation Sfil solution Southern blot specific spheroplasts sterile strains switch techniques tion Tris-HCl trypanosome tube V/cm vector vector DNA yeast yeast artificial chromosomes