Experiment no 3: Preparation of Solutions, Buffers and Stocks for DNA Isolation.

Solutions
A solution is a homogeneous mixture of two or more substances. A solution may exist in any
phase. A solution consists of a solute and a solvent. The solute is the substance that is dissolved
in the solvent. The amount of solute that can be dissolved in solvent is called its solubility. For
example, in a saline solution, salt is the solute dissolved in water as the solvent.
Preparation of Solutions
You prepare a solution by dissolving a known mass of solute (often a solid) into a specific
amount of a solvent. One of the most common ways to express the concentration of the solution
is M or molarity, which is moles of solute per liter of solution.
Molarity (M) = Wt (g) / Molecular weight (M.wt.)
Therefore;
Grams of solute required = [Mol wt. of solute X Concentration (M) X Volume in ml ]/1000

• Sometimes it's necessary to adjust the pH of a solution. To do this, add enough water to
dissolve the solute. Then add an acid or base solution dropwise (usually a hydrochloric
acid or HCl solution for acid or sodium hydroxide or NaOH solution for a base) to reach
the desired pH. Then add more water to reach the mark on the glassware. Adding more
water won't change the pH value.
Buffer Solutions
Buffers are solutions that resist change in pH on dilution or on the addition of small amounts of
acids or alkali.
Buffer solutions are water-based liquids that include both a weak acid and its conjugate base.
Because of their chemistry, buffer solutions can keep pH (acidity) at a nearly-constant level even
when chemical changes are taking place. Buffer systems occur in nature, but they are also
extremely useful in chemistry.
Buffer Action
So, how does a buffer work? Let’s take the example of a mixture of acetic acid (CH 3COOH) and
sodium acetate (CH3COONa). Here, acetic acid is weakly ionized while sodium acetate is almost
completely ionized. The equations are given as follows:
CH3COOH H+ + CH3COO–
CH3COONa Na+ + CH3COC–
To this, if you add a drop of a strong acid like HCl, the H+ ions from HCl combine
with CH3COO– to give feebly ionized CH3COOH. Thus, there is a very slight change in the pH
value. Now, if you add a drop of NaOH, the OH– ions react with the free acid to give
undissociated water molecules.
CH3COOH + OH– CH3COO– + H2O
In this way, the OH– ions of NaOH are removed and the pH is almost unaltered.
Uses for Buffer Solutions
In organic systems, natural buffer solutions keep pH at a consistent level, making it possible for
biochemical reactions to occur without harming the organism. When biologists study biological
processes, they must maintain the same consistent pH; to do so they used prepared buffer
solutions. Buffer solutions were first described in 1966; many of the same buffers are used
today.
To be useful, biological buffers must meet several criteria. Specifically, they should be water
soluble but not soluble in organic solvents. They should not be able to pass through cell
membranes. In addition, they must be non-toxic, inert, and stable throughout any experiments for
which they are used.
Buffer solutions occur naturally in blood plasma, which is why blood maintains a consistent pH
between 7.35 and 7.45. Buffer solutions are also used in:
• fermentation processes
• dying fabrics
• chemical analysis
• calibration of pH meters
• DNA extraction
What Is Tris Buffer Solution?
Tris is short for tris(hydroxymethyl) aminomethane, a chemical compound which is often used in
saline because it is isotonic and non-toxic. Because it has a Tris has a pKa of 8.1 and a pH level
between 7 and 9, Tris buffer solutions are also commonly used in a range of chemical analyses
and procedures including DNA extraction. It is important to know that pH in tris buffer solution
does change with the temperature of the solution.
How to Prepare Tris Buffer
Materials:
Calculate the amount of each item you need based on the molar concentration of the solution you
want and the quantity of buffer you need.
• Tris (hydroxymethyl) aminomethane
• EDTA
• Sodium dodecyl Sulphate (SDS)
• Sodium Acetate
• distilled deionized water
• HCl
Procedure:
 1. Start by determining what concentration (molarity) and volume of Tris buffer you want to
make. For example, Tris buffer solution used, varies from 10 to 100 mM.
2. Next, determine how many grams of Tris this is by multiplying the number of moles by
the molecular weight of Tris (121.14 g/mol). grams of Tris = (moles) x (121.14 g/mol)
3. Dissolve the Tris into the distilled deionized water, 1/3 to 1/2 of your desired final
volume.
4. Mix in HCl (e.g., 1M HCl) until the pH meter gives you the desired pH for your Tris
buffer solution.
5. Dilute the buffer with water to reach the desired final volume of solution.
Once the solution has been prepared, it can be stored for months in a sterile location at room
temperature. Tris buffer solution's long shelf life is possible because the solution does not
contain any proteins.
Stock Solution
A stock solution is a concentrated solution that will be diluted to some lower concentration for
actual use. Stock solutions are used to save preparation time, conserve materials, reduce storage
space, and improve the accuracy with which working lower concentration solutions are prepared.

Preparation of dilution from stock solutions

Use the law of conservation of mass to perform the calculation for the dilution:
CstockVstock = CdilutionVdilution
or C1 V1 = C2 V2
Dilution Example
As an example, say you need to prepare 100 milliliters of a 1mM solution from a 0.5 M EDTA
stock solution. Your first step is to calculate the volume of stock solution that is required.
Note: convert mM concentrations to M first.
C1 V1 = C2 V2
(0.5 M)(x ml) = (0.001 M)(100 ml)
x = [(0.001 M)(100 ml)]/0.5 M
x = 0.2 ml or (200ul) of stock solution
To make your solution, pour 0.2 ml of stock solution into a 100 ml volumetric flask. Dilute it
with solvent to the 100 ml line.
Avoid This Common Dilution Mistake
It's a common mistake to add too much solvent when making the dilution. Make sure you pour
the concentrated solution into the flask and then dilute it to the volume mark.
For dilution, we make up the remaining Volume with distilled water.
Do not, for example, mix 250 ml of concentrated solution with 1 liter of solvent to make a 1-liter
solution.

Calculations (to be written on Blank page of Practical book)

Formulas used to make solutions:

Grams of solute required = [Mol wt. of solute X Concentration (M) X Volume in ml ]/1000
First, we prepared Stock solution of Tris and EDTA separately
Preparation of 100mM Tris, 50ml and 100mM EDTA, 10ml)
1. 100mM Tris, (0.1M Tris) ; Mol wt. 121.14 g/mol
grams of Tris Base required = (121.14 x 0.1 x 50)/1000
= 0.6028g
0.6028g → 80ml dH20 → pH adjusted to 8 → make up volume to 50ml with distilled water

2. 100mM EDTA (0.1M EDTA), pH- 8.0, Mol wt. 336.21 g/mol
grams of EDTA required = (336.21 x 0.1 x 10)/1000
= 0.336g
Dissolve 0.336g EDTA in 7ml water → add 2 pallets of NaOH → EDTA with dissolve now → adjust pH to
8.0 → make up volume to 10ml with dd water.
3. 10% SDS , vol - 10ml
Weight 1g SDS and dissolve in 10ml distilled water in small 15ml falcon tube.

4. 3M Sodium Acetate, pH 5.2, Vol- 10 ml

Grams of Sod Acetate required = (82.04 x 3 x 10)/1000
= 2.461g
Dissolve 2.461g sod acetate in 7ml distilled water in small 15ml falcon tube → place falcon tube
in warm water for 10min to dissolve → then set pH to 5.2 with Glacial acetic acid → make up vol
to 10ml with distilled water

How we made working solution of Lysis buffer that is, TE buffer (10mM Tris, 1mM EDTA)?
First we Calculated volume to be taken from stock solution Using formula C1V1 = C2V2
Vol of Tris required
0.1M x V1 = 0.01M x 50ml
=> V1 = [0.01x50]/0.1 = 5ml
Vol of EDTA required,
0.1M x V1 = 0.001M x 50ml
=> [v1 = [0.001 x 100]/0.1 = 0.5ml
From Stocks, Add 5ml Tris and 0.5ml of EDTA in big 50ml falcon tube and make up volume to 50ml
with distilled water. Label the tube as TE buffer for bacterial lysis