Practical Molecular Biology Lab. 01 October 18, 2020

Students should able to know and explain:

Ѽ What is the Solution?

Ѽ What is the dilution technique?

Ѽ How can you use the prefixes to modify units?

Ѽ What is the Purpose of preparing the volume and conc. of solution?

Ѽ How is Solution preparation done?

Ѽ How can you measure the length and the weight of DNA?

Metric Prefix:

A metric prefix is a shorthand notation used to denote very large or vary small

values of a basic unit as an alternative to expressing them as powers of 10. Basic

units frequently used in the biological sciences include meters, grams, moles,

and liters. Because of their simplicity, metric prefixes have found wide

application in molecular biology. The following table lists the most frequently

used prefixes and the values they represent.

Units of Measurement and Conversion factors:

Translating a measurement expressed with one metric prefix into an

equivalent value expressed using a different metric prefix is called a conversion.

These are performed mathematically by using a conversion factor relating the two

different terms. A conversion factor is a numerical ratio equal to 1.

Problem: a) There are approximately 3 * 109 bp per human haploid genome.

What is this number expressed as kilobase pairs (kb)?

b) Convert 0.03 mg into ng?

c) How can you measure the length and the weight of DNA?

A T (1Base Pair) = 617 Dalton.

G C (1Base Pair) = 618 Dalton.

1 pg. (one picogram) of DNA = 106 kb= 109 bp.

Human haploid genome has 3pg, how?

The distance between two successive nucleotide sequence is 3.4 A˚ = 0.34 nm.

DNA length in one human haploid genome is one meter (1.02m) how?

Dilution Technique:

Dilution technique is used for expression of solution concentration that is

commonly used in biology laboratory. Dilution provides a simple accurate method

for changing the concentration of solution.

Dilution factor= total number of unit volumes.

Example 1: to make 1:20 dilution of NaCl solution and water, we add 1 unit

volume of NaCl solution with 19 unit volumes of water.

Example 2: Dilute DNA sample 2:500 using unit volumes of (µl)s.

ANSWER/ mix 2 µl of the DNA sample with 498 µl of solvent.

Note: the unit vol. could be anything from a liter, micro liter, nano liter….etc.

Practical Molecular Biology Lab. 01 October 18, 2020

A serial dilution is a series of simple dilutions, in which the Final Dilution

Factor, (FDF) = Last DF

Q) Make three step serial 1:10 dilutions from RNA containing solution, by

taking out 10 µl from the original sample?

A) In order to make three 1:10 dilutions using 10 µl, we add to each of the sample

tubes 10 µl of the dilute from the previous tube with 90 µl of diluent (solvent).

The Dilution factor in sample (1) = 1:10, sample (2) =1:100, and sample (3)

=1:1000, so the final sample in this question is 1000 times diluted more than the

original RNA sample.

Dilutions can be calculated using the C1V1 = C2V2 equation, one approach is

to use the equation C1V1 = C2V2, where C1 is the initial concentration of the stock

solution, V1 is the amount of stock solution taken to perform the dilution, C2 is

the concentration of the diluted sample, and V2 is the final, total volume of the

diluted sample.

Dilution can be defined as the mass of original substrate or previously

deposited track melted, divided by the sum of the combined mass of substrate and

added material melted.

In chemistry and biology, the dilution ratio is the ratio of solute to solvent.

It is often used for simple dilutions, one in which a unit volume of a liquid material

of interest is combined with an appropriate volume of a solvent liquid to achieve

the desired concentration.

The diluted material must be thoroughly mixed to achieve the true dilution.

Often, a worker will need to change the concentration of a solution by

changing the amount of solvent. Dilution is the addition of solvent, which

decreases the concentration of the solute in the solution. Concentration is the

removal of solvent, which increases the concentration of the solute in the solution.

(Do not confuse the two uses of the word concentration here!)

In both dilution and concentration, the amount of solute stays the same. This

gives us a way to calculate what the new solution volume must be for the desired

concentration of solute.

Because this quantity does not change before and after the change in

concentration, the product MV must be the same before and after the

concentration change. Using numbers to represent the initial and final

conditions, we have M1V1 = M2V2 as the dilution equation. The volumes must

be expressed in the same units.

Practical Molecular Biology Lab. 01 October 18, 2020

Preparation of Solution (Volume & Concentration):

is a homogenous mixture that consisting of two or more substances, one

of this is (solute) in more time is (solid) and is dispersed or diffuse through other

substances which is called (solvent) in more time is (liquid) like water.

Types of Solution:

A. is prepared by dissolving an exact weighted amount of

pure (100%) substances that diluted in a known volume of a liquid (solvent)

and stored in volumetric flask in proper condition.

B. is prepared in high degree of accuracy with exact

concentration and preserved in special condition in which each time a known

amount take for preparation of required solution, this type of solution is more

useful in biological researches.

C. is a type of solution that using for preserve living

samples in their physiological conditions, (for example, Normal Saline).

D. is a common solution that can resist any change in the pH of

its environment.

means is the relative proportion of solute and solvent in a

solution.

The concentration of solution may be expressed in any one of the following

ways:

1. is a solution in which 1gm molecular weight of substances

(solute) is dissolved in solvent and completed to 1L.

Molarity=

Molarity=

No. of grams (weight) = = ( ) g of substance weighted and dissolved

in amount of distilled water and then

completed to volume we needed in

volumetric flask.

Mole

Volume

Weight (g) / Molecular Weight (g/mole)

Desired volume (ml) / Volume (L) 1000

M * V (ml) * M.Wt.

1000ml (1L)

2. is a solution in which 1gm equivalent weight of substances

(solute) is dissolved in solvent and made up to 1L.

Normality (N) =

Normality =

No. of grams (weight) = =

Normality =

3. is the solution containing known of the

(solutes), may be expressed according to the (physical prosperity) of the

materials:

which calculated by [Weight by Weight (W/W %)].

which calculated by [Weight by Volume (W/L %)].

which calculated by [Volume by Volume (L/L %)].

4. is the amount of substance per unit mass of solvent,

commonly given in units of mol/kg.

5. This is a way of expressing very dilute

concentrations of substances. Just as per cent means out of a hundred, so parts

per million or ppm means out of a million. Usually describes the

concentration of something in water or soil. One ppm is equivalent to 1

milligram of something per liter of water (mg/l) or 1 milligram of something

per kilogram soil (mg/kg).

( ) g of substance weighted and

dissolved in amount of distilled

water and then completed to

volume we needed in volumetric

flask.

Equivalent

Volume

o

l

u

m

e

Weight (g) / Equivalent

Weight

Desired volume (ml) / Volume (L)

1000Weight

N * V (ml) * Equivalent Weight

1000ml (1L)

Special gravity * % * 1000

Equivalent Weight

Practical Molecular Biology Lab. 01 October 18, 2020

Centrifugation is a method of separating molecules having

different densities by spinning them in solution around an axis (in a centrifuge

rotor) at high speed. It is one of the most useful and frequently employed

techniques in the molecular biology laboratory. Centrifugation is used to collect

cells, to precipitate DNA, to purify virus particles, and to distinguish subtle

differences in the conformation of molecules.