Lab Protocols

Buffer pK a Neutral form HCl salt Na + salt H 3 PO 4 / NaH 2 PO 4 (pK a1 ) 2.12 98.0 - 120.0 Glycine (pK a1 ) 2.34 75.07 111.5 - Citric acid(pK a1 ) 3.13 Acetic acid 4.75 60.05 - 82.0 Citric acid(pK a2 ) 4.76 192.1 - 294.1 MES 6.15 195.2 - 217.2 Cacodylic acid 6.27 H 2 CO 3 / NaHCO 3 (pK a1 ) 6.37 62.01 - 84.01 Citric acid(pK a3 ) 6.40 Bis-Tris 6.50 209.2 245.7 - ADA 6.60 190.2 - 212.1 Bis-Tris Propane (pK a1 ) 6.80 282.4 318.9 - PIPES 6.80 302.4 - 325.3 ACES 6.90 182.2 218.7 - Imidazole 7.00 68.1 104.5 - BES 7.15 213.2 - 235.2 MOPS 7.20

This media is prepared as two separate solutions to avoid precipitation: Solution A: NaCl 1.0 g MgSO 4 7H 2 O 0.2 g K 2 SO 4 1.0 g CaCl 2 2H 2 O 40 mg NaNO 3 2.5 g FeSO 4 7H 2 O 10 mg Gaffron micronutrients 1.0 ml Deionized water to 500 ml Solution B: K 2 HPO 4 0.5 g Na 2 CO 3 4.03 g NaHCO 3 13.61 g Deionized water to 500 ml After autoclaving, mix Solution A and Solution B asceptically. Final pH 9.4-9.8. Gaffron micronutrients: H 3 BO 3 3.1 g MnSO 4 4H 2 O 2.23 g ZnSO 4 7H 2 O 0.22 g (NH 4 ) 6 Mo 7 O 24 4H 2 O 0.088 g Co(NO 3 ) 2 6H 2 O 0.146 g VOSO 4 6H 2 O 0.054 g Al 2 (SO 4 ) 3 K 2 SO 4 2H 2 O 0.474 g NiSO 4 (NH 4 ) 2 SO 4 6H 2 O 0.198 g Cd(NO 3 ) 2 4H 2 O 0.154 g Cr(NO 3 ) 3 7H 2 O 0.037 g Na 2 WO 4 2H 2 O 0.033 g KBr 0.119 g KI 0.083 g Deionized water to 1 L

100% Steinberg’s Solution (AKA 5xMR) (makes 2 liters) 40 ml NaCl stock solution (17% w/v NaCl) 20 ml KCl stock solution (0.5% w/v KCl) 20 ml CaCl2 stock solution (0.5% w/v CaCl2) 20 ml MgSO4 stock solution (1.025% w/v MgSO4) 1 ml gentamycin stock solution (4% w/v gentamycin) 2.4 g HEPES 1899 ml ddH2O Adjust pH to 7.4 - 7.6 and store in refrigerator. Usually, you will make 2-4 liters at a time. To make the required stock solutions,

Treat tads with ~1.5 ml PTU prior to stage 30-32 to prevent pigmentation. 0.3% PTU 0.3 g Phenylthiocarbamide (Sigma # P-7629, also called Phenylthiourea) 100 ml 20% Steinberg’s solution Some of the PTU will not dissolve. Shake solution prior to use to dissolve some of the precipitate (put “shake well” on label). Store in refrigerator.

100 ml 10X MBS stock 1.25 ml 4% gentamycin (1 g gentamycin sulfate in 25 ml ddH2O) 893 ml ddH2O 7 ml autoclaved 0.1M CaCl2 (11.1 g CaCl2 / 100 ml ddH2O) Adjust pH to ~7.4. Sterile filter prior to use. Make fresh weekly and store in refrigerator.

Modified Barth’s Saline Operating Solution (MBS) (makes 1 liter) 100 ml 10X MBS stock 500 ul 1% phenol red (pH indicator, if needed) 10 ml pen/strep antibiotic cocktail 5 ml 4% gentamycin (1 g gentamycin sulfate in 25 ml ddH2O) 0.2 g Ethyl 3-aminobenzoate methanesulfonic acid salt (C9H11NO2*CH4SO3) 7 ml autoclaved 0.1M CaCl2 (11.1 g CaCl2 / 100 ml ddH2O) Fill to 1000 ml with ddH2O. Adjust pH to ~7.4. Sterile filter immediately prior to use. Make fresh each week and store in refrigerator. NOTE: the anesthesia amount may need to be decreased for some tads.

2% Agar (for agar dishes) 2 g Agar 100 ml ddH2O Microwave to dissolve the agar, lightly swirling the mixture periodically. Turn off the room light to see the solution better; it will boil over if not watched carefully. Stop the microwave when the solution starts to boil up in the flask and swirl gently. Be careful while swirling the solution as it may unexpectedly boil over due to the added kinetic energy. Use orange heat resistant gloves to handle the flask. Once the solution has cooled a bit, microwave again, stopping when it boils. Repeat this several times – the more boiling, the less bubbles in your finished agar dishes. Pour the hot mixture into petri dish lids and let it set. Store the dishes in an air tight container in the fridge. Make new every two weeks.

1X Tadpole Anesthesia (0.05%) 10 ml 10X Tadpole Anesthetic 90 ml 20% Steinberg’s solution or 0.05g Ethyl 3-aminobenzoate methanesulfonic acid salt (C9H11NO2*CH4SO3) 100 ml 20% Steinberg’s solution Adjust pH to ~7.4.

use this anesthesia to anesthetize adult frogs before dissecting out testes, and also for 1X dilution. 10X Tadpole Anesthesia (0.5%) (AKA Adult Anesthetic, MS222, Tricane) 0.5 g Ethyl 3-aminobenzoate methanesulfonic acid salt (C9H11NO2*CH4SO3) 100 ml 20% Steinberg’s solution Initially the solution is very acidic. Be careful adjusting the pH, as it tends to jump quickly past pH 4.5. Our meter has trouble reading anesthesia and will never truly stabilize. Once the “S” symbol appears on meter, use pH paper to adjust pH to ~7.4.

We use this to anesthetize tadpoles before injections, etc. 1X Tadpole Anesthesia (0.05%) 10 ml 10X Tadpole Anesthetic 90 ml 20% Steinberg’s solution or 0.05g Ethyl 3-aminobenzoate methanesulfonic acid salt (C9H11NO2*CH4SO3) 100 ml 20% Steinberg’s solution Adjust pH to ~7.4.

Culture Media (makes 100 ml) 10 ml Fetal Bovine Serum (FBS) 1 ml pen/strep antibiotic cocktail (AKA Fungibact) 1 ml Embryo Extract solution (EE) 28 ml ddH2O 60 ml L15 Adjust pH to ~7.4. Sterile Filter prior to using; do not autoclave.

Dilute to 1X to yield zebrafish embryo rearing solution. This embryo medium contains antibiotics for fish that are susceptible to diseases (like transgenic and fluorescent fish). For the standard embryo medium without antibiotics, see 60X E3 stock. 20X E2 stock (makes 2 liters) 35 g NaCl 1.5 g KCl 0.82 g KH2PO4 4.8 g MgSO4 0.24 Na2HPO4 2000 ml ddH2O Autoclave (optional) and store in the refrigerator. Make the following 3 solutions. When diluting to 1X E2, combine 50 ml 20X E2, 2 ml CaCl2 solution, 2 ml of the NaHCO3 solution, and ddH2O to 1 liter. Add antibiotic diluted 1:500 in1X E2. 1. Add 14.5 g CaCl2 in 200 ml ddH2O, autoclave, and refrigerate. 2. Add 6 g NaHCO3 to 200 ml ddH2O, autoclave, and refrigerate. 3. Make a 60 mg/ml penicillin, 100 mg/ml streptomycin stock, aliquot and store at -20o C. Dilute 1:500 in 1X E2 for use.

Serum Free Culture Media (makes 100 ml) 1 ml pen/strep antibiotic cocktail (AKA Fungibact) 0.1 g Albumin, bovine serum (fatty acid free, Sigma# A8806) 39 ml ddH2O 60 ml L15 Adjust pH to ~7.4. Sterile Filter prior to using; do not autoclave.

Reaggregation Solution with 0.05% Anesthesia (makes 1 liter) 0.56 g Tris Base 0.32 g MgSO4 (7H2O) 6.81 g NaCl 0.05 g KCl 1.47 g CaCl2 (dihydrate) 1000 ml ddH2O 0.5 g Ethyl 3-aminobenzoate methanesulfonic acid salt (C9H11NO2*CH4SO3) Phenol Red (a few drops to indicate pH) Adjust pH to ~7.4. Sterile Filter prior to using; do not autoclave.

Low [Ca++] Reaggregation Solution with 0.05% Anesthesia (makes 1 liter) 6.81 g NaCl 0.05 g KCl 1.47 g CaCl2 (dihydrate) 1.91 g HEPES 1000 ml ddH2O 0.5 g Ethyl 3-aminobenzoate methanesulfonic acid salt (C9H11NO2*CH4SO3) Phenol Red (a few drops to indicate pH) Adjust pH to ~7.4. Sterile Filter prior to using; do not autoclave.

Disaggregation Solution (makes 1 liter) 6.81 g NaCl 0.05 g KCl 0.56 g Tris Base 0.117 g EDTA 1000 mL ddH2O Phenol Red (a few drops to indicate pH) Adjust pH to ~7.4. Sterile Filter prior to using; do not autoclave.

Embryo Extract Solution (EE) (makes 100 ml) 60 % L15 10 % FBS (Fetal Bovine Serum) 30 % ddH2O 1 % FB (Fungibact; Antibiotic antimycotic) 1. Remove jelly coat and membrane from embryos (stage 23-30) and count (you will need the number of embryos later). 2. Place embryos in tissue grinder, drawing off as much liquid as possible. 3. Add ~1 squirt of EE media. 4. Homogenize embryos (~ 50 plunges). Push down slowly and pull up quickly. 5. Transfer to 2 centrifuge tube and increase volume to ~ 30 mls using EE media or about 1/2 full. Balance both tubes. 6. Spin at 15K, 4C for 90 mins in SS-34 rotor. 7. Three layers appear. Bottom = Crude, Middle = Liquid, Top= Milky lipid layer. Carefully pipette middle layer. Avoid the top layer because it clogs the filter. 8. Add EE media such that the final concentration is 1 ml / 1 embryo. 9. Sterile filter.

PBT 100 ml 1X PBS g BSA (bovine serum albumin Cohn Analog (Sigma# A1470)) 100 ul triton Make fresh each use. Can be stored in refrigerator for no more than 24 hours.

0.4 M PO4 Buffer Solution (makes 500 ml) 5.25 g NaH2PO4 * H2O 43.3 g Na2HPO4 * 7H2O (or 22.94 g Na2HPO4) 500 ml ddH2O Adjust pH to ~ 7.4.

0.1 M PO4 Buffer Solution (makes 400 ml) 100 ml 0.4 M PO4 buffer solution 300 ml ddH2O Adjust pH to ~ 7.4.