Drosophila_injections - 1 Drosophila germline...

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Unformatted text preview: 1 Drosophila germline transformation Nicolas Gompel, March 2005 This protocol is designed for P-element based transformation (Spradling and Rubin, 1982) and also works for other retrotransposons such as Hermes (Horn and Wimmer, 2000). Preparing the DNA (for P-element based constructs) • Make a midiprep of the construct of interest and of the helper plasmid pπ25.7 ∆2-3 wc (Spradling and Rubin, 1982) (e. g. using a Qiagen ® Plasmid PuriFcation Kit). Avoid phenol puriFcation. Resuspend DNA at 1 µg/µl. • Injection mix Construct plasmid (1 µg/µl ) 10 µl Helper plasmid (1 µg/µl) 9 µl ¡iltered (0.2 µm) food dye 1 µl Alterantively (but I havenʼt tested it myself), the construct can be injected in ¢ies that have a stable source of transposase (progeny of w; ∆2-3 98B; + x w; +; +. The w; ∆2-3 98B is available from the Bloomington center, #...). In this case the injection mix would be: Construct plasmid (1 µg/µl ) 19 µl ¡iltered (0.2 µm) food dye 1 µl • Vortex, spin 15 min at max speed. Carefully harvest 17 µl from the top and transfer to a new tube. Use 1 µl or less to Fll the needles (capillaries have an internal Flament and a droplet of mix added at the open end will migrate to the tip of the needle). Sometimes, a tiny air bubble forms at the tip of the needle and will make injection difFcult. It will go off 30 min or so after Flling the needle. Once Flled, the needles can be stored in a humid chamber (on a bar of plasticin in a Petri dish with a wet Kimwipes in the lid). They can be kept at 4˚C for a few days. Flies A yw mutant strain is best suited for this protocol, since it allows detection of white rescue phenotypes or expression of a ¢uorescent protein in the adult eye. Also, the y phenotype provides an extra security that red-eyed ¢ies are not contaminants . These ¢ies are used both as a source of embryos for the injection and as a backcross stock to amplify the transformants. ¡lies are kept in bottles on standard medium. Transfer about 300 ¢ies per egg-lay cage (figure on the left) 2-3 days before the Frst injection, to allow them acclimate to these egg-lay conditions. Egg-lay caps should be changed at least twice a day when not injecting. The population should be kept large enough to permit easy female virgining for backcrosses. 2 Set-up The injection set-up consists of two parts: an inverted microscope equipped with a 20x lens and a micromanipulator, and an air-pressure injecting device (e.g., Narishige IM-300 Microinjector) connected to the needle holder. Bright Feld or Nomarski diascopy are most appropriate to monitor injections. The micromanipulator can be attached to the stage or not, but if it is not, it should be Frmly attached to the bench....
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Drosophila_injections - 1 Drosophila germline...

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