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.

Popping Buffer

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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.[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:

I have written a simple code in LabView V9.1 with the download link:[8]

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:

[math]\displaystyle{ C_s=C_b*V_b/V_m }[/math] in moles

Now to calculate mass to be measured:

[math]\displaystyle{ M=M_w*C_s*V_s }[/math] 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

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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.

• 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.