Short Protocol for Microinjection in X. Tropicalis

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Supplies:
diamond pencil
hand pipetteman
injection needles (program 1)
tips
epi tube
pipette
tropicalis injection dishes
1008 petri dishes
injection substance
plastic tip box
plastic beaker (for liquid waste)
tweezers
pestle

Reagents:

1/9X MR
3% Ficoll in 1/9X MR, autoclaved

3% Cysteine in 1/9x MR, pH 7.8-8.0
1/9X MR + GENT (1ml gent/200ml MR)
10X MBS high salts
0.1X MBS PH 7.8 - 8.0 Important: check it the same day
1X MBS + 0.1% BSA, filter sterilized
Milli-q H20

Night Before:

____ Collect one male, three females

____ Prime females w/ 10 units hCG

____ Boost male w/50 units hCG

____ Set up incubator temp. (18-28°C)

Injection Day:

____ FIRST THING IN MORNING: Boost females w/200 units hCG (Time 0)

____ Pull necessary needles for experiment

____ Make cysteine (3% in 1/9 MR, Ph=7.8-8.0)

____ Set up injection substance (dilutions)

____ Bring down:

____ inj. substance (on ice)

____ cysteine

____ After Benzocaining male, remove testes and put in 1X MBS + 0.1% BSA

____ Squeeze females for eggs in plates previously coated with 1X MBS + 0.1% BSA.

____ Release sperm from testes using ball crusher (pestle).

____ Pour sperm on eggs, mix well with pestle WAIT 3 MINUTES.

____ Start fertilization by flooding with just enough 0.1X MBS to cover eggsImportant: PH 7.8 - 8.0 check it the same day

____ WAIT 10 minutes

____ Cysteine embryos for 5 min or until done.

____ Rinse 3 times with 0.1X MBS, then 2 times with 3% ficoll in 1/9 MR. Leave embryos in 3% ficoll in 1/9 MR.

____ Put injection substance into needle using pipette tip and pipette.

____ Break needles using tweezers, test w/oil 2nl ≅ 8 hash marks at top mag. For injections, remove media above embryos so that surface tension keeps embryos in place.

____ Label dishes, after injections are done add ficoll REST ONE HOUR in 22-28°

____ Change solution to 1/9x MR + GENT, put in 25°C or desired temp.

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

Buffers (From Early Development of Xenopus Laevis, Laboratory Manual, Sive, Grainger, Harland, Cold Spring Harbor Laboratory Press)

MBS (Modified Barth's Saline)
Two solutions: 0.1 M CaCl2 and 10X MBS salts.

0.1 M CaCl2= 11.1 g/liter, Autoclave and store aliquots at -20ºC or 4ºC.

10X MBS salts

880 mM NaCl
10 mM KCl
10 mM MgSO4
50 mM HEPES (pH 7.8)
25 mM NaHCO3
Adjust final pH ti 7.8 with NaOH, autoclave.

Prepare the final solution mixing 100 ml of 10X salt solution with 7 ml of 0.1 M CaCl2, and adjust the volume to 1 liter with distilled water. This is MBS 1X.

1X MBS + 0.1% BSA
Make 1X MBS and add 0.1% (w/v) of BSA, filter sterilized. Check pH: 7.8-8.0.

0.1X MBS
Dilute 1X MBS 10 times with distilled water, check pH: 7.8-8.0.