# User:Pranav Rathi/Notebook/OT/2012/10/01/Buffer preparation for DNA overstretching & unzipping experiments

< User:Pranav Rathi‎ | Notebook‎ | OT‎ | 2012‎ | 10‎ | 01

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.

## Contents

### Popping Buffer

This is a buffer we used for unzipping DNA experiments. It is funny but true 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.[1]

Sodium phosphate

monobasic: Sodium phosphate monobasic is H2Nao4P, it helps the PH-level by taking or giving OH- and H+ ions.[2]

dibasic: Sodium phosphate dibasic is HNa2O4P, it also helps PH-level maintenance. [3]

NaCl: Sodium chloride also helps with PH-maintenance. [4]

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.[5]

H2O: Primary solvent.[6]

D2O: Primary solvent. Either one can be used depending on the experiments.[7]

#### 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 mass of chemicals from the bottle are based on the stock volume, but the final concentration in the buffer volume will be based on the volume which is mixed into the buffer to make final buffer;

• EDTA: 10mL is mixed.
• Sodium phosphate:

mono: 1.9mL is mixed

di: 8.1mL is mixed(this is to keep the PH at 7.5)

• NaCl: 1.25mL is mixed
• Tween20: 1mL is mixed
• H2O or D2O: 77.75mL is taken

The final volume is 100mL. Stock volume is completely based on my desire, so all these concentrations and volumes are needed for calculations of measured chemical mass from the bottles.

##### Calculations:

The math is following:

• Desired buffer concentration is Cb(M)
• Desired final-buffer volume is Vb(L)
• Callulated stock concentration is Cs(M)
• Volume mixed into the buffer is Vm(L)
• Mass written on the bottle is Mw(gm/mole)
• Stock volume Vs(L)
• Calculated mass to be measured is M(gm)

First to calculate the stock concentration:

${\displaystyle C_{s}=C_{b}*V_{b}/V_{m}}$ in moles

Now to calculate mass to be measured:

${\displaystyle M=M_{w}*C_{s}*V_{s}}$ in gm

Now this mass is measured on the scale and mix in the desired stock volume, then mix volume is taken from the stock to mix into final buffer to get the final concentration. All the calculation are given in the excel spreadsheet: {{#widget:Google Spreadsheet |key=0ApjWjFYiQdkfdENGTm5kckg5dTNfN01uQjh2YUVyZWc |width=500 |height=300 }}

##### Procedure
1. Weight the chemicals on the scale
2. Measure and mix the chemicals in the solvent and mix using vortex
3. Measure the volume and mix into the buffer
4. Store
```See the slide share for more information
```

Next buffer is PBS.

### PBS

PBS is Phosphate Buffer Saline.It is a buffer solution commonly used in biological research. It is a water-based salt solution containing sodium chloride, sodium phosphate, and, in some formulations, potassium chloride and potassium phosphate. The buffer's phosphate groups help to maintain a constant pH. The osmolarity and ion concentrations of the solution usually match those of the human body (isotonic). We use PBS to make Anti-digsolution for DNA-tethering.[9]

• The chemical used in POP are as follows:
Salt Concentration Concentration
(—) (mmol/L) (g/L)
NaCl   137 8.01
KCl   2.7 0.20
Na2HPO4 • 2 H2O   10 1.78
KH2PO4   2.0 0.27
pH   7.4  7.4

#### Making PBS

The simplest way to prepare a PBS solution is to use PBS buffer tablets (see slides). They are formulated to give a ready to use PBS solution upon dissolution in a specified quantity of H2O or D2O. They are available in the standard volumes: 100, 200, 500 and 1000 mL.

##### Procedure

Dissolve 1 table in 200mL of H2O or D2O.

Using these two buffers we make all other solutions we use to make DNA-unzipping and overstretching sample.

### Blocking Solution (BGB)

The purpose of blocking solution is to block exposed glass surfaces after binding anti-dig. Various kinds of casein are typically used, which I think evolves from the common practice of using non-fat dried milk (NFDM) in standard wet-lab protocols, such as western blotting. NFDM is predominantly casein, and so people use NFDM and casein interchangeably, usually ignoring the fact that differences in purity or kinds of casein could potentially impact a sensitive assay. Often it is imagined that casein is a regular soluble protein, but Dr.Koch found in the past that casein forms polydisperse micelles, probably. He doesn't know whether these polydisperse micelles are important for it's blocking capabilities, but he did find some references that said they are (small micelles fill gaps in big micelles). Brent Brower-Toland, being a good biologist, ignored the anlaysis paralysis of physicists and just ordered cheap good blocker from Bio-Rad, called "Blotting-Grade Blocker" [10] at Bio-rad.com. This worked very well and we continue to use it. Bio-Rad calls it "non fat dried milk," so I'm not sure if it's the same stuff you get at the supermarket. We'll call this BGB from now on (which can also read as "Brent's Good Blocker.")

• Why use BGB; blotting grade blocker: BGB is casein (α, β and κ) which is hydrophobic and like to be clustered in water. It is used to block the surface where anti-dig is not present (coat that part with casein). Casein make small hemisphericalballs (micelle [11]), these balls fillup the gaps around the anti-dig preventing the beads sticking to the surface.

#### Procedure

To make 5mg/ml BGB solution in popping buffer:

1. First wiight out 15mg of BGB poweder
2. Add 1.5 ml of 1x pop and mix by vortexing
3. Run through .2μM syringe filter using 3ml syringe.
4. Now store 5mg/ml BGB in 1x pop buffer (minus amount of protein that stuck to filter) at 3C.

This should be good for few weeks.

### Anti-Dig

Polyclonal sheep anti-digoxigenin from Roche [12]. This is shipped as a lyophilized powder. We always resuspend entire 200 microgram bottle with 1 ml of ice-cold PBS, and then make 20 microliter aliquots which are stored at -80C. An aliquot can be extracted from freezer, and diluted with 180 microliters of cold PBS.

#### procedure

1. Add 1mL of ice-cold PBS into 200μgm of Anti-dig and mix it.
2. Make 20μgm/ml of aliquots.
3. Nitrogen flash-freeze them and store them at -80C.
4. When use for sample; take out an aliquot from freezer and mix 180mL of cold PBS and store at +3C.