Dejellying embryos (Zorn lab)
Protocol submitted by VGP
Dejellying solution:
2% Cysteine-hydrochloride, pH 8 in 0.1xMBS, to remove the jelly coat from fertilised Xenopus eggs and embryos before they can be manipulated.
| Volume of 0.1xMBS | Cysteine | 5N NaOH |
|---|---|---|
| 50 mls | 1g | |
| 100 mls | 2g | |
| 200 mls | 4g |
Volume of 0.1xMBS Cysteine 5N NaOH 50 mls 1g 100 mls 2g 200 mls 4g
Dissolve the Cysteine hydrochloride (or L-Cysteine hydrochloride-1-hydrate) in ~3/4 the final volume of 0.1xMBS.
Adjust to ~ pH 8 with 5N NaOH.
Check that the pH is correct and then adjust the final volume with 0.1xMBS.
This solution will only last one day.
Dejelling embryos:
Wait at least one hour after fertilisation, any time after this embryos may be dejellied.
Decant the MBS off the embryos, leaving them semi-dry and stuck to the bottom of the dish.
Cover the embryos in the 2% cysteine solution, which will dissolve the jelly coats surrounding the eggs.
With occasional, mild agitation, the embryos will be liberated in 5-7 minutes.
It is important not to “swirl” eggs too vigorously when dejellying as this can cause axis duplications in some cases.
Once the jelly coat is dissolved, the cysteine solution must be removed immediately (it can be reused).
The embryos are then washed 3-4 times in a large volume of 0.1xMBS to remove residual cysteine.
If left in the cysteine solution too long, or not washed enough, the plasma membrane will be damaged and the embryos will die!