Meganuclease Microinjection Protocol

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This is a Short Protocol, and does not detail IVF and Microinjection techniques. For details of IVF and microinjection methods, see details in specific protocols.

Reagents:

1/9x MR

1X MBS+ 0.2% BSA pH=7.8-8.0

0.1X MBS pH=7.8-8.0

3% Ficoll in 1/9x MR, autoclaved
3% Cysteine in 1/9x MR, pH 7.6-8
1/9x MR + GENT

Night Before:

____ Collect one male, three females
____ Prime females w/ 10 units hCG
____ Boost male w/50 units hCG
____ Set up incubator temp. (18-28C)

Injection Day:

____ Boost females w/ 200 - 250 units hCG (frogs start laying in 3-4 hours)
____ Make cysteine
____ Set up injection substance (dilutions)

MN Reaction
10X Buffer	2 µl	2 µl	2 µl	2 µl	2 µl
Plasmid(800ng total)
Scel enzyme	2 µl	2 µl	2 µl	2 µl	2 µl
H20 to 20 µl
Injection Order

Bring to injection station:

____ Injection substance (on ice)
____ Cysteine

Time	Other Things to Prepare
0 min ___Start MN Digest @ 37°C 2 min ___Place male in Benzocaine 8 min ___Dissect testes into 1X MBS+0.2% BSA 12 min ___Squeeze Eggs(coated dishes) 15 min ___IVF testis on eggs - 3 min 17 min ___Flood with 0.1X MBS - 10 min 27 min ___Cysteine 35 min ___Rinse(2 times 0.1% MBS, 2 times 3% Ficoll) 40 min ___MN Reaction to ice. Inject Inject 2 nl of MN reaction - 80 pg DNA	__Benchcoat for male dissection __tools for dissection __500 µl 1X MBS+0.2%BSA Ficoll and 1/9XMR ready __Coat injection dishes with 1X MBS+0.2%BSA __Label microinjection dishes __Pull needles __Aliquot Ficoll into injection dishes __Clean dissection tools

Label dishes; after injections are done, add ficoll.

REST ONE HOUR at 28°C

Change solution to 1/9x MR +/- GENT

4nl ≅ 10 hash marks 2nl ≅ 8 hash marks 1nl 6 ≅ hash marks