By Vilhelm A. Bohr (auth.), Lotte Bjergbæk (eds.)
Current wisdom of the mechanisms that keep watch over DNA fix has grown considerably during the last years with know-how advances corresponding to RNA interference, complicated proteomics and microscopy in addition to excessive throughput monitors. The 3rd variation of DNA fix Protocols covers a number of elements of the eukaryotic reaction to genomic insult together with fresh complex protocols in addition to commonplace options utilized in the sphere of DNA fix. either mammalian and non-mammalian version organisms are coated within the ebook, and lots of of the strategies may be utilized with merely minor adjustments to different structures than the only defined. Written within the hugely winning Methods in Molecular Biology? sequence layout, the chapters comprise the type of precise description and implementation suggestion that's an important for buying optimum leads to the laboratory.
Thorough and intuitive, DNA fix Protocols, 3rd variation provides professional counsel for DNA fix, recombination, and replication.
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Using primers specific to a target gene of interest, usually 500 bp apart, templates are PCR amplified twice, being gel purified each time, 96 targets at a time. As the primers also encode T7 primer sequences the amplified PCR products are then used for an in vitro transcription reaction to produce dsRNA, which is then purified, diluted, and aliquoted into stock plates ready for the screening experiment. 5. Equilibrate a water bath to 50 °C. 6. Slice out the area of the gel containing the amplified PCR product, by observing under UV transilluminator, and transfer into a pre-labeled microfuge tube or 96-deep-well plate.
Subsequently this screening method was utilized to develop different types of HTP assays to study various cellular functions (1). We have used this screening method and the cell viability assay combined with DNA-damaging agents to identify genes essential for cell survival after MMSinduced DNA damage (2). RNAi libraries for the Drosophila 18 D. R. Bishop Fig. 2. Work flow for high-throughput screening with dsRNA library. Robotics are used to realiquot dsRNA from 96-well stock plates into 384-well experimental plates.
Elegans mutants with increased/decreased sensitivity to RNAi in various tissues can also be obtained from the CGC. 3. C. elegans can be treated with compounds by casting it directly into the nematode growth media, adding it to nematode culture dish seeded with bacterial food or by soaking the nematode directly in the compound. Casting the compound directly in NGM ensures an even drug concentration throughout the plate but may affect growth of the bacteria as well and hence the efficiency of the RNAi.
DNA Repair Protocols by Vilhelm A. Bohr (auth.), Lotte Bjergbæk (eds.)