Not signed in (Sign In)

Welcome, Guest

You have reached the Lom Lab Protocols Resource, where lab protocols are stored, annotated, and discussed.

Please note that you must apply for an account and/or sign in before you can post a new protocol or comment on an existing one. You do not need to sign in to browse the protocols, however.

If you are interested in posting a new protocol, please be sure to read the Before You Post notice to ensure that our protocols remain standard. Thank you, and enjoy your visit!

 
Lom Lab Home

Vanilla 1.1.2 is a product of Lussumo. More Information: Documentation, Community Support.

    • CommentAuthorjuruble
    • CommentTimeJun 14th 2007 edited
     

    BT-Fix
    Warning: Do not make BT-Fix alone the first time.


    0.52 g NaH2PO4 * H2O
    2.28 g Na2HPO4

    200 ul of 0.12M CaCl2 (1.3 g CaCl2 / 100 ml ddH2O or 1.76 g CaCl2-H2O/100 mL ddH2O)

    8g Sucrose
    8 g Paraformaldehyde (Extremely Toxic – Hood, Gloves, Mask, Coat required)

    This recipe is for 200 mL of BT fix.

    Put on lab coat and gloves before making the solution. Add 170 mLs of Millipore water to a fix beaker. Place beaker on a stir plate, use a fix stir bar. Put all ingredients except paraformaldehyde into the beaker. 

    Place the stirplate under the hood with a fix beaker on it.  Turn the heat on to about 60 degrees celsius.    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.  Add the paraformaldehyde to the beaker, rinsing the powder off your scoop with the liquid in the beaker.

    When powders are dissolved (about 20-30mins), 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 water (and stir) if the total volume is too low.

    Let the solution cool completely to room temperature.  Filter the liquid through Millipore filter using vacuum suction.  pH this solution to ~7.4 using the fix electrode on the pH meter (make sure to connect this electrode to the meter). Check the solution volume in a graduated cylinder, add water if needed so that the total volume is 200 mLs. Aliquot the solution into 15 ml tubes and store in freezer.

    • CommentAuthorkalayman
    • CommentTimeJun 10th 2010
     

    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.