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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Page 15
... Incubate overnight at 37°C to generate spheroplasts. (9) Discard solution in a ventilated fume hood and rinse the samples several times with 0.25 M EDTA, pH 8.0. Discard the rinse solution in the fume hood. (10) Add 1–2 vol of digestion ...
... Incubate overnight at 37°C to generate spheroplasts. (9) Discard solution in a ventilated fume hood and rinse the samples several times with 0.25 M EDTA, pH 8.0. Discard the rinse solution in the fume hood. (10) Add 1–2 vol of digestion ...
Page 16
... 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) Store samples in ...
... 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) Store samples in ...
Page 34
... at 3000g at 4°C. Discard the supernatant. (4) Resuspend the cells by pipetting in 1 ml of Sorbitol-EDTA-3mercaptoethanol, then incubate at room temperature 5 min. (5) Add 40 pil of Zymolyase 100T solution (or 50 34 Ken Dewar et al.
... at 3000g at 4°C. Discard the supernatant. (4) Resuspend the cells by pipetting in 1 ml of Sorbitol-EDTA-3mercaptoethanol, then incubate at room temperature 5 min. (5) Add 40 pil of Zymolyase 100T solution (or 50 34 Ken Dewar et al.
Page 35
... incubate at 37°C for 30 min. (6) Calculate the spheroplasting efficiency by withdrawing an aliquot (5-10 ul) of the cell solution to a microscope slide, then (at 20–40X magnification) determine the percentage of cells that lyse ...
... incubate at 37°C for 30 min. (6) Calculate the spheroplasting efficiency by withdrawing an aliquot (5-10 ul) of the cell solution to a microscope slide, then (at 20–40X magnification) determine the percentage of cells that lyse ...
Page 36
... Incubate for 1 hr with agitation (~100 rpm). Calculate spheroplasting efficiency as already described. (6) Collect the cells by a 10-min centrifugation at 3000g at 4°C. Carefully remove the supernatant, as the cell pellet can be very ...
... Incubate for 1 hr with agitation (~100 rpm). Calculate spheroplasting efficiency as already described. (6) Collect the cells by a 10-min centrifugation at 3000g at 4°C. Carefully remove the supernatant, as the cell pellet can be very ...
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