Troubleshooting for Early Cold Shock Gynogenesis
My females won't lay eggs at the correct time .
1. You made up the boost wrong; double check your calculations.
2. They were improperly injected. Either some of the boost leaked out or you didn't inject completely into the dorsal lymph sac.
3. Some females may carry specific mutations that delay their ovulation; we have given 1.5X normal boost (in same volume) to induce the females to ovulate at the correct time. However, this was an established trend we'd noted after injecting this group of females several times over.
4. Some females are just stubborn.
I came in to do the experiment and my females had dumped all their eggs.
1. You made up the prime wrong and used too much HCG. Double check your calculations.
2. Your females haven't been used in a really long time (years) and just dumped all their old eggs.
My haploid dish contains all diploids.
1. You forgot to remove the lids from the Petri dishes before irradiation.
2. Your UV bulbs are burned out or your crosslinker is broken.
My haploid dish contains some diploids
1. A piece of tissue debris was aliquotted into the dish and some of the sperm escaped irradiation. Your experiment isn't useless; continue as you normally would.
I have little to no fertilization in ALL of my dishes.
Your males were nearly sterile.
You left the sperm sitting out too long and it began to die.
Your sperm is too dilute.
In the dishes from one female, I have little to no fertilization.
1. The female's eggs are not of good quality. This sometimes occurs with virgin animals, immature animals, old animals, animals that have not been used in a really long time, animals that haven't been properly rested, and sick animals. Generally the eggs will come out stringy or will have irregular shapes when viewed microscopically if they are not good quality.
I have little to no fertilization in my haploid and cold shock dishes, but my WT outcross dishes are fine.
1. Too much UV irradiation to the sperm. Double check your parameters. Your instrument may need calibrating.
2. Males' sperm may be weak. It will hold up to in vitro fertilization, but cannot tolerate UV irradiation.
All my cold shock embryos died almost immediately or the recovery was really low.
1. The media was too cold or too warm. Be sure to check the temperature before putting it on the embryos.
2. The timing of the cold shock application was not correct. Be sure you are using the “assembly line” method when adding and removing media.
3. The eggs were of poor quality.
My tadpoles have been developing for four or five days and now look “fuzzy”.
1. Tadpoles are developing an infection because they haven't been properly cared for or are too crowded. Decrease the density of the dishes to less than 50 and treat with a media that has a higher salt concentration.
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