In vitro fertilization of Xenopus laevis (Conlon lab)

Protocol submitted by VGP from Conlon lab protocols [1]

link to protocol document [2]

in vitro Fertilization of Xenopus laevis Protocol

Modified 4/15/2005

Egg Collection

1) Allow female to lay eggs in 1X MMR

2) Collect eggs in a small beaker in 1X MMR

Testis Preparation

1) Remove testis from male into 1X MBS

2) Remove blood, fat, and debris while in 1X MBS

3) Place testis in L15 media

4) Wash testis in 1X MBS before mincing (Perform all of the following as quickly as possible)

5) Mince testis with razorblade on a petri dish cover

Fertilization

1) Remove all but 20 ml of 1X MMR from beaker with eggs

2) Quickly add 40 ml 0.1X MBS to beaker (making it roughly 33% MMR)

3) Let eggs settle and quickly remove as much liquid as possible

4) Add 1ml 0.1X MBS to testis and resuspend sperm

5) Add the 1ml of sperm to the beaker with the eggs

6) Swirl a few times (I actually swirl the eggs and sperm once every few minutes until fertilized)

7) The eggs should fertilize and rotate within 10-15 minutes @ RT or 20-30 min @ 17°C