Biotech Glossary | Bioinformatics | Lab Protocol | Notes | Malaysia University |
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X-Ray Mutagenesis of Drosophila |
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| Overview |
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| This protocol describes the general procedure for creating mutations in the DNA of Drosophila by exposure to X-rays. Irradiation of cells with X-rays creates double strand breaks (DSBs) in DNA. Mutations introduced in the DNA of germ line cells (sperm) are propagated by mating the exposed males to virgin females. The progeny of this cross can be mated to each other so that a percentage of the subsequent offspring will have two copies of the same mutant allele. These flies can be examined for phenotypic changes. | |
| Procedure |
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1. Collect hundreds of 0 to 2 day old adult Drosophila males. 2. Allow them to age for 2 days at 25°C. 3. Anesthetize them with ether, or equivalent, and aliquot the flies into groups of 50 each. 4. Place each group of 50 into a gelatin capsule (Electron Microscopy Sciences, Size #000) that has been perforated with a 23-gauge needle. 5. Place the capsules in a petri dish on ice and incubate for 5 min (see Hint #1). 6. Place the dish in the X-ray machine and expose the flies to a 4000 Rad dose of X-rays (115 kV at 5 mA; see Hint #2 and Hint #3). 7. Immediately transfer the flies into a bottle with plenty of yeast and fly food. Allow the irradiated flies to recover for a few hours. 8. Introduce the males to an equal number of 3 day-old virgin females and allow the flies to mate (see Hint #4). 9. After a few hours, transfer the flies into fresh bottles on successive days. 10. On the fourth day, discard the flies (see Hint#5). 11. Collect flies from each of the bottles. Examine them for genetic markers and phenotypic changes (see Hint #6). |
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| Solutions |
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| This bioProtocol does not use any solutions | |
| BioReagents and Chemicals |
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Ether |
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| Protocol Hints |
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1. Do not allow the capsule or the flies to become wet. 2. CAUTION! Wear protective gear and take all protective actions to minimize exposure to X-ray energy. This step requires specialized equipment. Please consult experienced personnel before attempting this procedure. 3. Alternatively, males can be placed in empty vials, and the vials can be placed on their sides in the X-ray machine followed by exposure to the 4000 Rad dose. 4. 30 females is a good number as some of the irradiated males will not survive the exposure. 5. Starting with 400 males (8 irradiated groups), 10,000 to 20,000 progeny flies can be easily obtained for screening. 6. Often, males and females will carry mutations that allow for easy identification and selection of flies with certain traits (e.g., red eye pigment, "curly" wings, etc.). |
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