I worked with Pranav to make some H2O and D2O buffers for the shotgun DNA mapping experiment. We use a buffer that we call Popping Buffer for our tethering experiment. The name comes from the fact that this buffer was used during Koch’s (and potentially others’) experiments that involved “popping” bound proteins off DNA while unzipping. The buffer is (final concentration):

• 50mM NaCl
• 50mM Sodium Phosphate (which is a mixture of dibasic and monobasic sodium phosphate)
• 10mM EDTA
• 0.02% Tween-20

We made two 100ml amounts of solution in D2O and H2O respectively. And the buffers were concocted from separately made solutions in each water type:

• 4M NaCl was made as a solution in both D2O and H2O
• 500mM Sodium Phosphate (monobasic) in D2O and H2O
• 500mM Sodium Phosphate (dibasic) in D2O and H2O
• 100mM EDTA in D2O and H2O

From some Popping Buffer we made BGB (Blotting Grade Blocker) in both D2O and H2O at a concentration of 5mg/ml (about 15ml volume). And we also made aliquots of anti-dig in PBS (from only H2O, since we aliquot 20ul amounts and then add 180ul of Popping buffer when it is experiment time).