![]() Homogenization of sugar beet leaves for the purpose of extracting and isolating DNA for subsequent PCR amplification requires a sufficient amount of good quality nucleic acid yield (>1ng). => 98% of the samples achieved good yields. DNA Yields from sugar beet leaf material using the Geno/Grinder and a 96 well micro titer plate kit (NucleoSpin Multi-96 Plant, Machery & Nagel). The in vitro reproduction of linear DNA with GenomiPhi (GE Healthcare) allows further DNA analyses with small sample sizes.įigure 1. With a pool composition of 8 different samples the utilized DNA amount is reduced to 0.125ng/individual plant so that with 100ng DNA, the selection of DNA pools with 800 different target sequences becomes possible. The selection of the mutant population is carried out with DNA pools. A further reduction of the DNA amount is possible. For a routine PCR 1ng of highpure DNA is used. Only 2% of the samples did not yield an adequate amount of DNA. Afterwards the samples can be stored long-term at -20☌.Īpproximately 40% of the samples yielded between 1ng DNA. DNA quantification is carried out by photometry and gel electrophoresis. After mixing, filtration, several rinse steps and drying the samples are finally eluted. Following lysis, the centrifuged samples are transferred into micro titer plates (round wells). After grinding the tissue to a fine powder the samples were charged with 300 μl C1-buffer and homogenized in the Geno/Grinder for 30 seconds at a setting of 1000 (1000 strokes min-1). This type of sampling allows a flexible configuration of the individual samples. For this, four mounts (plastic blocks) were produced in-house, each holding 24 Eppendorf vessels, and clamped into the Geno/Grinder. The plant tissue (0.5-1 g) was freeze dried in a 2ml Eppendorf vessel and then ground in the Geno/Grinder with two metal balls at a setting of 1000 (1000 strokes min-1) for 2 x 1 minute. (NucleoSpin Multi- 96 Plant, Machery & Nagel, Düren). The effective detection of point mutation occurs via hetero duplex formation between DNA molecules of wild type and mutant.ĭuring the first project year, DNA from 2688 individual plants was isolated by using a 96- well micro titer plate kit. ![]() To establish a TILLING platform with automated DNA extraction and PCR amplification of target genes, nucleic acid extraction of consistent yield and good quality is necessary. ![]() TILLING technology is a new and very effective method of reverse genetics for the production and identification of loss/gain of function-mutations of commercially valuable genes without genetic modification. First a representative range of EMS (Ethyl methane sulfonate)-mutants is produced from 2000-5000 M2 plants and identified through TILLING. ![]() Within the framework of a TILLING project (Targeting Induced Local Lesions In Genomes) nucleic acids from leaf material are to be isolated from potential mutants. Application Notes SP011: Tissue Homogenization Cell Lysis ![]()
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