20ml glass scintillation vial
Small stir bar
* P-phenylenediamine ( EMD Chemicals Inc. Cat# PX0730)
Carbonate-Bicarbonate Buffer (see below)
Wrap a glass scintillation vial with foil and drop in a small stir bar. (PPD is light-sensitive.)
With a 10 ml pipet add 9 mls of glycerol to the vial.
With the 1000 l Pipetman add 1ml of 1X PBS.
Place on stirrer and begin mixing.
Weigh out 10 mg of p-phenylenediamine on the Mettler balance.
PPD is toxic. Wear gloves and dont inhale it.
Add the PPD to the vial and stir untill it is all in solution (1-2 hrs.). The medium should appear almost colorless to a slight tint of yellow. If it is an intense yellow or orange color the PPD is most likely contaminated and will have background staining.
pH the mounting medium to a pH of 8.0-9.0 using the Carbonate-Bicarbonate buffer. pH paper of range 6.5-10.0 should be used to check the pH of the medium after addition of 12 drops of the Carb-Bicarb buffer and stirring. Additional drops of buffer are added untill the desired pH is reached.
Aliquot the mounting medium and store at -70oC.
* Flakes of PPD are large and should be crushed
Carbonate- Bicarbonate Buffer
Make up a 0.2M solution of anhydrous sodium carbonate (2.12g/100ml)
Make up a0.2M solution of sodium bicarbonate (1.68g/100ml)
Take 4 mls of A + 46 mls of B and bring up to 200 mls with DH2O. The pH will be 9.2.
*Note: If the PPD is contaminated or goes bad ( turns a brown color ) it will stain DNA, so each preparation should be tested. Check by looking at mitotic cells to be sure that chromosomes are not stained.