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.
H_20: Primary solvent
D_2O: Primary solvent. Either one can be used depending on the experiments.