For the past few days, Pranav’s been hard at work collecting DNA tweezing data. The first set of experiments revolve around DNA overstretching (which I’ll explain later, but you should feel free to google it). We’re planning on analyzing force differences between DNA in D2O and H2O through overstretching. Hopefully eventually we’ll be working towards DNA unzipping, but that is less likely given our time constraints (graduation!!). Regardless there are a lot of interesting studies to be done here.
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).