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From 2012.igem.org

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Contents 1 PCR with Phusion High-Fidelity DNA Polymerase 1.1 Tube preparation 1.2 Cycling instructions 2 Préparation of LB medium and LB Agar: 3 Gel extraction 4 In vitro fecondation of Xenopus tropicalis 4.1 2 or 3 days before the IVF 4.2 D day 4.3 Euthanasia of the male

PCR with Phusion High-Fidelity DNA Polymerase

Tube preparation

Put items in this order:

Component	50µl reaction	Comments
H2O	32
5x Phusion HF Buffer	10
10mM dNTPs	1
Primer FW	2	Primers have to be at 10µM
Primer RV	2	Primers have to be at 10µM
Template DNA	1
DMSO (optional)	1,5	recommended for GC-rich amplicons < 20kb
Phusion DNA polymerase	0,5

Cycling instructions

Cycle step	Temperature	Time	Cycles
Initial denaturation	98°C	4min	1
Denaturation	98°C	20s	30
Annealing	Lower Tm of primers	30s
Extension	72°C	30S/kb
Final extension	72°C	10min	1
	4°C	hold

Préparation of LB medium and LB Agar:

=> LB Agar : -18,5g LB Agar -300ml H2O

=> LB medium : -6g LB broth -300ml de H2O

Autoclaved at 250°C

Gel extraction

1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel under UV light.
2. Weight the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel.
3. Incubate at 50°C for 10 min, until the gel slice has completely dissolved.
4. The color of the mixture have to be yellow, otherwise add 10 µl of 3M sodium acetate.
5. Ass 1 gel volume of isopropanol to the sample and mix.
6. Place a spin column in a provided 2 ml collection tube.
7. To bind DNA, apply the sample to the column and centrifugat for 1 min. Discard the flow-through and place the column back into the same tube.
8. To wash, add 0,75 ml of buffer PE to the column and centrifugate for 1 min.Discard the flow-through and place the column back into the same tube.
9. Centrifugate the column in a 2 ml Collection tube for 1 min 17,900xg (13,000 rpm).
10. Place the column into a clean 1,5 ml microcentrifuge tube.
11. to elute DNA, add 15 µl of water to the column and centrifugate for 1 min.

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In vitro fecondation of Xenopus tropicalis

2 or 3 days before the IVF

Pre-injection of frogs (males et females)
100µl of hCG (0,1UI/µl, do a dilution by 10 of the commercial solution)

D day

Test the injector before beginning
Inject the 100µl hCG (1UI/µl) in the morning
Isolate the animals : an animal by box

Prepare :
Timer
Methylcellulose (put at room temperature)
Boxes special for injections
Materiel of injection
Capillaires ad hoc with oil
Solutions to inject (add 1µl of blue or red of Nile)
Solution L15/FCS :
- boxes (1/femelle) of Pétri (d=10 cm) lined of L15/FCS (500µl)
- 2 tubes 1,5mL with 250µl of L15/FCS (one for each testicle)
Pestles in plastic cleaned to grind the testicles
MMR 0,1X
MMR 0,05X
MS-222 0,2%

Euthanasia of the male

Put the male in a vat with MS-222 0,2% and look forward to his non response of pinches. Put it out and process of the dissection to extract the two testicles.
For that, dispose the animal: the dorsal face against the dissection board. Pinch the skin in the middle of the belly and do a small incision with scissors; from this point, perform skin incisions shown in the diagram.

Fold the flaps and skin incision in the muscular wall avoiding severing the abdominal vein to avoid hemorrhage. Both gonads are located in the ventral face of kidneys.
Both gonads are located in the ventral surface of the kidneys, they cover more or less and in corpora lutea (fat mass).By extracting them from the abdominal cavity, we can extract the testicles attached to them. The male's testicles are two oval organs, smooth, yellow-pale as can be seen in the photograph below.