Skip to Main Content

INFORMATION FOR

Meganuclease Microinjection Protocol

Word

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 Buffer2 µl2 µl2 µl2 µl2 µl
Plasmid(800ng total)
Scel enzyme2 µl2 µl2 µl2 µl2 µl
H20 to 20 µl
Injection Order

Bring to injection station:

____ Injection substance (on ice)
____ Cysteine


TimeOther 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