User:Pranav Rathi/Notebook/OT/2012/10/01/Buffer preparation for DNA overstretching & unzipping experiments
We primarily use popping buffer and PBS for DNA-unzipping and overstretching experiments. All the other buffers consists these two. This page discusses the chemicals and process used in preparation of these buffers.
This is a buffer we used for unzipping DNA experiments. It is funny but thrue that it's called "popping buffer" because when DNA binding proteins are present they are "popped" off the DNA when it is unzipped. Popping buffer or POP is the main solution used to prepare the samples. It can be made in H2O or D2O. The primary purpose of this buffer is to maintain the PH-level and stabilize the DNA or DNA-protein complex in the solution. The popping buffer I use is 1X; which means the salt (NaCl) concentration is whatever it is in standard POP. By doubling or tripling it, 2X or 3X POPs can be made.
- The chemical used in POP are as follows:
EDTA: Ethylenediamineteraacetic acid disodium salt dehydrate. EDTA usually binds to metal cation, such as mg+2 ions and ca+2 ions. This makes DNA-protein complex more stable and prevent protein or enzymes to cut the DNA.
monobasic: Sodium phosphate monobasic is H2Nao4P, it helps the PH-level by taking or giving OH- and H+ ions.
dibasic: Sodium phosphate dibasic is HNa2O4P, it also helps PH-level maintenance.
NaCl: Sodium chloride also helps with PH-maintenance.
Tween 20: Polyethylene glycol sorbitan monolaurate is a detergent which prevents non-specific antibody binding and to saturate binding sites on surface. Basically it helps with DNA-tethering which uses anti-dig and dig bonding.
H2O: Primary solvent
D2O: Primary solvent. Either one can be used depending on the experiments.
Making Popping buffer (POP)
Let's say I need 100mL of 1X POP in H2O or D2O with the following concentration of chemicals:
- Desired Buffer-Volume: 100mL
- EDTA: 10mM in 100mL of buffer volume
- Sodium phosphate: Total concentration is 50mM in 100mL of buffer volume
Mono is 19% of 50mM + Di is 81% of 50mM =50mM in 100mL of buffer volume
- NaCl: 50mM in 100ml of buffer volume.
- Tween20: .02% of 100ml
To get these desired concentrations I convert the moles into grams and prepare the following stock in the following way:
It is always good to keep the chemicals in stock, so the measured quantities are based on the stock volume, but the final concentration in the buffer volume will be same based on the following percentages;
- EDTA: 10% of 100ml buffer volume=10mL
- Sodium phosphate:
mono: 2% of 100ml buffer volume = 1.9mL
di: 8% of 100ml buffer volume = 8.1mL (this is to keep the PH at 7.5)
- NaCl: 1.25% of 100ml buffer volume =1.25mL
- Tween20: 1% of 100ml buffer volume = 1mL
- H2O or D2O: 77.75% of 100ml buffer volume =77.75mL
The final volume is 100mL