Xip Labis egg extracts are useful as sources of cytoplasmic and organelle material for in vitro reconstitution of cellular events such as nuclear assembly and nuclear protein import. Of course, before you can use egg extracts, you first need to obtain the eggs from female frogs. Hi, my name's Marie Cross and I'm a graduate student in Dr.Maureen Power's lab in the Department of Cell Biology at Emory University in Atlanta, Georgia.
Today we're gonna show you how to obtain and D jelly eggs from mature opus lavis females. This procedure includes the following steps, priming frogs for egg, laying with pregnant marere, serum gonadotropin inducing egg, laying with human chorionic gonadotropin, and sorting out any bad eggs or cytes. So let's get started.
To produce eggs, mature female frogs are first primed with pregnant mare serum gonadotropin. To begin the priming procedure, prepare a tank containing a hundred millimolar sodium chloride to house frogs. After injection, prime frogs should be kept at a density of approximately one frog per liter.
Optimal egg production is maintained by housing frogs at 18 degrees Celsius. Using a 27 gauge needle attached to a one mil syringe, inject frog subcutaneously into the dorsal lymph sac with 0.5 mils of 200 units per mil. Pregnant mare serum gonadotropin.
Return prime frogs to the tank containing a hundred millimolar sodium chloride. After two to three days, induce egg laying by injecting frogs with 0.5 mils of a hundred units per mil. Human chorionic gonadotropin again inject subcutaneously into the dorsal lymph sack.
After injection, place each frog in five liters of a hundred millimolar sodium chloride. It is best to put each frog in its own container as the quality of eggs varies from frog to frog. About 16 to 22 hours after the human chorionic gonadotropin injection, remove the frog to a clean water tank and pour the eggs into a beaker.
It is advisable to observe the frogs for one to two days for signs of infection at the site of injection. At this stage, a transparent jelly coat surrounds each egg, which prevents them from touching each other to de jelly eggs, rinse eggs once and 2%L cysteine pH H eight, then continued to incubate in a hundred bills of 2%L cysteine solution per frog for approximately five minutes. At room temperature, gently swirl the eggs periodically after five minutes.
Pour the cysteine solution off of the deje eggs and wash them three times in a 0.25 x modified ringer solution. To remove debris, pour eggs into a hundred millimeter glass Petri dish working under a dissecting microscope. Remove the bad eggs from the Petri dish with a pasture pipette.
Mature xip eggs have a darkly pigmented half on top and a light half on the bottom. A white pinpoint size spot indicative of oocyte maturation should be visible in the center of a darkly pigmented hemisphere of the mature egg. A good egg is one with even on modeled pigmentation.
Anything without this white spot is an oocyte and should be removed. Any eggs that are white enlarged or not uniformly pigmented should also be removed. A particularly bad egg will appear as a white puffy ball with a boundary between the hemispheres entirely obscured wash sorted eggs two to three times by pouring egg lys buffer on them, and then carefully pouring the buffer off, minimizing the number of eggs that are spilled out.
Now eggs can be lysed and extracts can be prepared. We've just shown you how to obtain and de jelly eggs from mature xenopus lavis females. Be sure to check out our other two videos which show you how to prepare crude egg extracts as well as how to reconstitute in vitro nuclear assembly using fractionated egg extracts.
So that's it. Thanks for watching and good luck with your experiments.
