BT-Fix (to fix tadpoles)
BT-Fix Warning: Do not make BT-Fix alone the first time.
0.26 g NaH2PO4 * H2O
1.14 g Na2HPO4
100 ul of 0.12M CaCl2 (1.3 g CaCl2 / 100 ml ddH2O or 1.76 g CaCl2-H2O/100 mL ddH2O)
4 g Sucrose
4 g Paraformaldehyde (Extremely Toxic – Hood, Gloves, Mask, Coat required)
This recipe is for 100 mL of BT fix.
Put all ingredients except paraformaldehyde into a 100 ml graduated cylinder (if you are doubling the recipe, you need a graduated cylinder that goes up to at least 200 ml). The 0.12M CaCl2 can be mixed in advance and kept in the refrigerator. Fill the graduated cylinder to almost 100 ml with ddH2O (you need to leave room for the rest of the ingredients, so don't fill it all the way to 100 ml. You can always add more later).
Place the stirplate under the hood with a fix beaker on it. Place a fix thermometer into the fix beaker. Pour the mixture you just made into the fix beaker. Put a fix stir bar into the beaker and turn on the stirplate. Turn the heat on to about midway between 30 degrees and 100 degrees. Put on lab coat, goggles, and gloves as it heats. Move the balance into the hood to prepare to measure out the paraformaldehyde with a fix scoop. Use a large weigh boat to weigh the paraformaldehyde to decrease the likelihood that any will spill onto the balance. When your solution is between 50-60 degrees Celsius, turn off the heat and add the paraformaldehyde, rinsing the powder off your scoop with the liquid in the beaker.
When powders are dissolved, add NaOH (solution or pellets) slowly until the solution clears. Each pellet takes about 30 seconds to 1 minute to dissolve, and for 200 ml of fix, it takes about 3-4 pellets to clear the liquid. Keep the NaOH pellets closed in between adding pellets or they may harden into a solid mass. After the solution clears, you can add more ddH2O (and stir) if the total volume is too low.
Let the solution cool completely to room temperature. Filter the liquid through filter paper in a fix funnel into another fix beaker. pH this solution to ~7.4 using the fix electrode on the pH meter (make sure to connect this electrode to the meter). Aliquot the solution into 15 ml tubes and store in freezer.
Solution must be aliquoted into 15 ml tubes, not 50 ml tubes. After being thawed the fix is only good for a maximum of two weeks, so freezing it in smaller aliquots ensures that we won't waste a large tube of fix.
0.26 g NaH2PO4 * H2O
1.14 g Na2HPO4
100 ul of 0.12M CaCl2 (1.3 g CaCl2 / 100 ml ddH2O or 1.76 g CaCl2-H2O/100 mL ddH2O)
4 g Sucrose
4 g Paraformaldehyde (Extremely Toxic – Hood, Gloves, Mask, Coat required)
This recipe is for 100 mL of BT fix.
Put all ingredients except paraformaldehyde into a 100 ml graduated cylinder (if you are doubling the recipe, you need a graduated cylinder that goes up to at least 200 ml). The 0.12M CaCl2 can be mixed in advance and kept in the refrigerator. Fill the graduated cylinder to almost 100 ml with ddH2O (you need to leave room for the rest of the ingredients, so don't fill it all the way to 100 ml. You can always add more later).
Place the stirplate under the hood with a fix beaker on it. Place a fix thermometer into the fix beaker. Pour the mixture you just made into the fix beaker. Put a fix stir bar into the beaker and turn on the stirplate. Turn the heat on to about midway between 30 degrees and 100 degrees. Put on lab coat, goggles, and gloves as it heats. Move the balance into the hood to prepare to measure out the paraformaldehyde with a fix scoop. Use a large weigh boat to weigh the paraformaldehyde to decrease the likelihood that any will spill onto the balance. When your solution is between 50-60 degrees Celsius, turn off the heat and add the paraformaldehyde, rinsing the powder off your scoop with the liquid in the beaker.
When powders are dissolved, add NaOH (solution or pellets) slowly until the solution clears. Each pellet takes about 30 seconds to 1 minute to dissolve, and for 200 ml of fix, it takes about 3-4 pellets to clear the liquid. Keep the NaOH pellets closed in between adding pellets or they may harden into a solid mass. After the solution clears, you can add more ddH2O (and stir) if the total volume is too low.
Let the solution cool completely to room temperature. Filter the liquid through filter paper in a fix funnel into another fix beaker. pH this solution to ~7.4 using the fix electrode on the pH meter (make sure to connect this electrode to the meter). Aliquot the solution into 15 ml tubes and store in freezer.
Solution must be aliquoted into 15 ml tubes, not 50 ml tubes. After being thawed the fix is only good for a maximum of two weeks, so freezing it in smaller aliquots ensures that we won't waste a large tube of fix.
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