Download Notch Signaling: Methods and Protocols by Hugo J. Bellen, Shinya Yamamoto PDF

By Hugo J. Bellen, Shinya Yamamoto

Notch Signaling: tools and Protocols specializes in molecular, biochemical and bioinformatics elements of Notch signaling. Chapters examine the Notch interactome, post-translational alterations of Notch, ligand binding assays, easy methods to determine proteolytic cleavage and transcriptional pursuits. Written within the hugely winning Methods in Molecular Biology series structure, chapters comprise introductions to their respective themes, lists of the required fabrics and reagents, step by step, conveniently reproducible laboratory protocols and key pointers on troubleshooting and keeping off identified pitfalls.

Authoritative and practical, Notch Signaling: tools and Protocols is a beneficial contribution to hundreds of thousands of labs and hundreds of thousands of scientists who pursue this learn sector with vigour.

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All crosses are performed with single males. Asterisk represents the EMS-induced mutation. cl = l(2) cl (cell lethal mutation) 16. Day 18: At a minimum of 10 days after setting the crosses, start collecting F1 males for phenotypic scoring. Isolate flies of interest and backcross them to ensure genetic transmission as well as amplification of the mutation into a stock (F1) (Fig. 2). 17. Day 28: At a minimum of 10 days after setting the F1 crosses, start collecting F2 males for confirmation of genetic transmission of the pre-observed phenotype.

Contamination by EMS cannot be measured. Thus, EMS should be handled with extreme caution. Any experiments with EMS must be conducted in a closed chemical hood wearing a lab coat, safety glasses, a mask, and double layers of gloves, to avoid accidental tearing. The chemical hood should be lined with a diaper to absorb EMS spills. The highest risk of contamination occurs mainly when the EMS solution is prepared and distributed. In the case of a contaminating spill, one should wipe the spot with Kimwipes, previously dipped in inactivating solution, available in a beaker in the hood.

Starved flies will efficiently uptake EMS through feeding the next day. 3. Day 7: Prepare the chemical hood for EMS mutagenesis according to safety instructions described in Note 3. Briefly, do not forget to use appropriate protective clothing and double layers of gloves. There must be chemical waste disposal beakers with inactivating solution previously prepared (see Note 3), where all equipment that has been exposed to EMS, such as syringes, fly bottles, and Whatman filter paper, may be discarded and decontaminated.

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