UNIVERSITY OF AGRICULTURAL SCIENCES,

GKVK, BANGALORE – 560 065

PRACTICAL MANUAL
MBB-504: TECHNIQUES IN MOLECULAR BIOLOGY-I: (1+2)

PREPARED BY
DR. NAGESHA, N
ASSISTANT PROFESSOR
MRS. POORNIMA, R.
ASSISTANT PROFESSOR
DR. ANITHA PETER
PROFESSOR
 
DEPARTMENT OF PLANT BIOTECHNOLOGY
COLLEGE OF AGRICULTURE, UASB, GKVK, BANGALORE-65
2023
 CONTENTS

Sl. No. Date Particulars Page No. Signatur

e
1. Good Laboratory Practices and Preparation of Buffers
and Reagents
2. Principles of Centrifugation and Spectrophotometry
3. Growth of bacterial culture and preparation of
growth curve
4. Isolation of Genomic DNA from bacteria
5. Isolation of plasmid DNA from bacteria
6. Growth of lambda phage and isolation of phage DNA
7. Isolation and restriction of plant DNA
8. Quantification of DNA by (a) Agarose Gel
electrophoresis and (b) Spectrophotometry
9. PCR using isolated DNA
10. PAGE Gel electrophoresis
11. Restriction digestion of plasmid and phage DNA,
ligation, Recombinant DNA construction
12. Transformation of E. coli and selection of transformants
13. Principles of Chromatographic techniques
 TLC
 Gel Filtration Chromatography
 Ion exchange Chromatography
 Affinity Chromatography
14. Dot-blot analysis, Southern hybridization, Northern
hybridization, Western blotting and ELISA
15. Radiation safety and non-radio isotopic procedure
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EXERCISE NO.1: DATE:
GOOD LABORATORY PRACTICES, PREPARATION OF
BUFFERS AND REAGENTS
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a) GOOD LABORATORY PRACTICES:
Aim: To understand the good laboratory practices and general rules and regulations
Principle:
A laboratory is a workshop for researcher/scientist/students. Here research group does the
techniques for preparation of chemical substances and formulate new methods. One must know what
all procedures are involved in the experiment and what all types of equipment and chemicals are
required for it. Proper knowledge about the working principles of the types of equipment and the
nature of the chemicals are essential
“Students are expected to behave professionally at all times. Lab notebooks will be maintained and
graded in the lab. We will provide background and relevant information about the solutions,
preparation, procedure and related techniques”.
Objectives:
The objectives are as follows,
 Develop the basic techniques of a Stock solution preparation, Tissue culture Techniques etc,
 Develop critical thinking skills in the students.
 Practice accuracy in calculations and in writing scientifically.
 Encourage teamwork and accountability among the students.
 Encourage students to take charge of their learning.
 Learn the responsibilities associated with working in a group.
Instructions to work in the laboratory:
1. Lab work is always totally different from any kind of office work. Researcher should be punctual
and a researcher is not absolutely free from research thoughts at any time of the day.
2. Always the research results reflects researcher attitude towards work.
3. Irresponsibility leads to failure of work in the laboratory.
4. Always maintain neat and clean environment in the Laboratory.
5. Always keep all the chemicals, glassware and all lab belongings should be placed in correct
place.

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6. Keep working area clean and neat; never keep books, purse bags on the working table.
7. Don’t eat or drink or talk while working in the laboratory.
8. Always keep lab observation book, a pen or pencil, a laboratory coat, a mask, a lab slipper and
pair of gloves tom work in the laboratory.
9. Always record observations, results at a time. For any difficulty and clarifications ask laboratory
in-charge or concern course teacher.
10. Every time record single calculation in lab observation book and every step involved in the
procedure.
11. Proper planning of work is must to finish work in time.
12. Carefully and economically use reagents and other materials. As much as possible use small
quantities of reagents.
13. Be careful while using glass equipments, if it breaks bring it to the notice of class teacher.
14. Proper dispose all the waste liquids in the sink and run water in the sink for sometime by
opening water tap.
15. Don’t spill any chemicals in or on the lab equipment, if spills, clean the equipment soon after its
use.
16. All the electrical supplies must be plugged out if not used.
17. Don’t switch on the lights, fans, AC’s and computer systems unless if it is required.
18. All water taps should be tightly closed after use.
19. Save electricity, water and gas for future needs.
20. First aid box maintained in the laboratory for medical assistance for treating any injury or burns.
21. Always maintain good relationship with laboratory colleagues for their valuable support,
22. Always update with the recent trends and findings at least in the concerned research field.
Documentation: The Lab Notebook
Introduction:
Documentation in a lab notebook is an essential skill for any biotechnician. The Food and Drug
Administration's (FDA) handbook states, "if it isn't written down, it wasn't done." Documentation
details vary from lab to lab but it is always done for one or all of the following reasons:
 To record what an individual has done and observed
 To prove that a procedure was done correctly
 To adhere to, evaluate, and develop standard operating procedures (SOP)

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Even good lab work is worthless without documentation, and careful documentation can turn
an erroneous result or a failed procedure into a valuable learning experience by providing essential
details needed for trouble-shooting. Furthermore, in industry, laboratory notebooks are legal
documents. They are used to determine patent rights, product quality, liability, and verify the
accuracy of information. Notebooks are treated as if they might be used in a court of law at any time,
and you can, in fact, be called upon for questioning about your notebook in court.
An important part of this documentation process is to record what equipment and materials
were used, and to show that the equipment and materials were validated and used in the correct
manner.
Lab Notebook:
Each student will maintain a lab notebook from which the lab reports will be derived. Your lab
notebook will be graded. Each of the Biotechnology and Biology laboratory classes in the
Biotechnology Program will have different formats and rules in regards to your notebook and lab
report format. Be sure to pay attention to your instructor, syllabus and lab manual in regards to this.
This is no different than in a typical Biotechnology workplace.
General rules for writing good lab notebooks are:
 Write all parts of your lab in ink. Writing with pencil is forbidden in the lab. It's too easy for
unscrupulous people to erase data or errors that they don't like, at which point important details
about their work are lost. If you make an error, draw a single line through it and enter your
correction in clear and legible writing. If you discard data for any reason, you must justify your
decision to do so immediately and in writing.
 Write legibly. Remember, supervisors, will be reading your notebook, and if they cannot read
your writing, your work is essentially non-existent. If they cannot easily make out what you
have written, they can easily misinterpret an important detail about your work.
 Never cover information in your notebook with anything else or store information on a sheet of
paper separate from your notebook. Never fold a page into your notebook. It can easily be lost.
 If you tape materials such as a graph, a manufacturer‘s specification sheet, or instrument
readout into your notebook, tape all four sides. Then write "NWUI" ("No writing under
insert") on the tape, your initials, and the date.

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 Keep your records factual, concise, clear and complete in all aspects. Write down important
details that have a bearing on your results so that you can answer any questions that might be
asked of you about how you did your work.
General safety precautions in handling hazardous chemicals in the lab
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Purpose:
There are special approaches and precautions that must be taken in any biological laboratory. This
includes procedures for safe handling and storage of hazardous chemicals and biological. Also, the
special methods for setting up and following detailed protocols are emphasized, as well as methods
for recording and archiving results properly.
There are generally four routes to exposure to hazardous chemicals that you should keep in
mind while handling them:
 Inhalation: avoid by the use of fume hoods and masks
 Skin & eye contact: avoid by the use of lab coats, gloves, and goggles
 Ingestion: avoid eating or drinking in the lab or leaving the lab without removing gloves and
washing hands
 Injection: dispose of broken glass and needles properly
 The chemicals in powder or crystal form that you are likely to encounter on this course will
generally be irritants in one way or another, possibly causing irreversible effects on contact with
the skin or on inhalation, always wear gloves when weighing, dispensing and handling solutions.
Disposal of hazardous chemicals & biological materials:
The disposal of hazardous chemicals is subject to state and federal regulations, and is ultimately
overseen by the Environmental Protection Agency. Extremely toxic chemicals are regulated at low
levels, and less toxic chemicals can be disposed of through city sewer systems at higher levels.
Biological hazards should be contained in autoclave bags made of a high melting point plastic that
are sealed and autoclaved at high temperatures and pressures to completely kill any live organisms
Safe handling of instruments
Keeping the equipments in good conditions in the laboratory is prerequisite for all the
researchers. It is always advisable not to use/ handle the instruments unless knowing the instructions
for its proper use. If you find any difficulties in handling the equipments take necessary guidelines

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from the technical persons and report immediately to the concern person if instruments are not
functioning properly.

 Check the power connections properly and switch on the power followed by the instruments. It
is better to switch on the instrument 15 minutes prior to use.
 If any power fluctuation do not switch on the instrument.
 Care should be taken not to switch on power with a wet hand.
 Do not spill water inside the equipment particularly during centrifugation if so keep the
instrument open and wipe off immediately.
 Make sure that the required temperature is attained before working with theinstrument.
 It is advisable to close the lid tightly if there is any, before running the instrument.
 Some machines need to keep close and some are in open conditions depending on the need.
 After using the instruments/equipments, first switch off the machine and followed by the
power button.

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b) PREPARATION OF BUFFERS AND REAGENTS
-----------------------------------------------------------------------------------------------
Aim: To understand and learn about the preparation of buffers and reagents
A buffer solution (pH buffer or hydrogen ion buffer) is an aqueous solution consisting of
a mixture of a weak acid and its conjugate base, or vice versa. Its pH changes very little when a small
amount of strong acid or base is added to it. Buffer solutions are used as a means of keeping pH at a
nearly constant value in a wide variety of chemical applications.
A reagent is an integral part of any chemical reaction. A reagent is a substance or compound
that can facilitate a reaction, and they are used in most widely used tests.
Reagents trigger chemical reactions. This term encompasses organic substances that trigger
naturally occurring chains of reactions in the body but also include inorganic substances that can be
used in artificially triggered reactions. Reagents are commonly used to test for the presence of certain
substances, as the binding of reagents to the substance or other related substances triggers certain
reactions.
Buffers are aqueous systems that resist changes in pH when small amounts of acid or base are
added. Buffer solutions are composed of a weak acid (the proton donor) and its conjugate base (the
proton acceptor). Buffering results from two reversible reaction equilibria in a solution wherein the
concentration of proton donor and its conjugate proton acceptor are equal. For example, in a buffer
system when the concentrations of acetic acid and acetate ions are equal, addition of small amounts
of acid or base does not have any detectable influence on the pH. This point is commonly known as
the isoelectric point. At this point there is no net charge and pH at this point is equal to pKa .
pH = pKa + log [CH3COO–]
[CH3COOH]

At isoelectric point [CH3 COO– ] = [CH3 COOH] hence, pH = pKa

Recommendations for the setting of the pH value of a buffer
Temperature: Depending on the buffer substance, its pH may vary with temperature.
Titration: Generally, the pH value is set using NaOH/KOH or HCl. Slow addition of the acid or base
whilst stirring vigorously avoids local high concentrations of H+ or OH– ions.

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Ionic strength: The setting of the ionic strength of a buffer solution (if necessary) should be done in
the same way as the setting of the pH value when selecting the electrolyte, since this increases
depending on the electrolyte used.
Buffer additives: If other components are added to the buffer (e.g. EDTA, DTT, Mg2+), changes in
the pH should also be expected and it should be retested.
pH meter control: Presently, accurate pH meters with a digital display are usually available for the
setting of the pH value of a buffer. The pH meter is calibrated using two pH standards which cover
the range of the buffer to be set. If there are any doubts about the precision of the device, this can
simply be resolved by standardising the pH meter using 50 mM phosphate buffer, which is then
diluted 10fold. The pH value should then be 0.2 pH units higher (Scopes 1994).
What makes a good buffer?
In 1966, Norman Good and colleagues set out to define the best buffers for biochemical systems
(Good et al. 1966). Good set forth several criteria for such buffers:
 A pKa between 6 and 8. Most biochemical experiments have an optimal pH in the range of
6–8. The optimal buffering range for a buffer is the dissociation constant of the weak acid
component of the buffer (pKa) plus or minus pH unit.
 Solubility in water. Biological reactions, for the most part, occur in aqueous environments,
and the buffer should be water-soluble for this reason.
 Exclusion by biological membranes. This is not important for all biochemical reactions.
However, if this is an important criterion for particular experiment, it is helpful to remember
that zwitterionic buffers (positive and negative charges on different atoms within the
molecule) do not pass through biological membranes. Examples of zwitterionic buffers
include MOPS and HEPES; Tris and phosphate buffers do not isomerize into zwitterions.
 Minimal salt effects: The buffer components should not interact or affect ions involved in
the biochemical reactions being explored.
 Minimal effects on the dissociation from changes in temperature and
concentration. Usually there is some change in the dissociation with a change in
concentration. If this change is small, stock solutions usually can be diluted without
changing the pH. However, with some buffers, changes in concentration have more effect on
dissociation, and stock solutions cannot be diluted without significantly affecting pH.

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 Minimal interactions between buffer components and critical reaction components: If
a complex forms between the buffer and a required cofactor, say a metal cation like zinc or
magnesium, your reaction also might be compromised. For example calcium precipitates as
calcium phosphate in phosphate buffers. Not only would any Ca2+-requiring reactions be
compromised, but the buffering capacity of the phosphate buffer also is affected.
Having excessive amounts of a chelating agent in the buffer for an enzymatically driven
reaction could cause problems (e.g., a high concentration of EDTA in PCR amplification).
Citrate is a calcium chelator, so avoid citrate buffers in situations where calcium
concentrations are critical.
 Chemical stability. The buffer should be stable and not break down under working
conditions. It should not oxidize or be affected by the system in which it is being used. Try
to avoid buffers that contain participants in reactions (e.g., metabolites).
 Light absorption. The buffer should not absorb UV light at wavelengths that may be used
for readouts in photometric experiments.
 Ease of Use. The buffer components should be easy to obtain and prepare.

General guidelines for preparation and storage of buffers and solutions:

The following guidelines should be followed when preparing buffers andsolutions.
1. It is generally easier to start by weighing dry components. Carefully weigh out the
desired amount of each component. Large amounts of components can be weighed
directly into the vessel in which they will be dissolved, e.g., measuring cylinder or
beaker.
2. For smaller amounts, use weighing boats and add each component sequentially once
the correct amount has been weighed out. Rinse weighing boats with distilled water
over the buffer vessel to ensure that all the substance weighed out has been added.
3. Measure liquid components using a measuring cylinder and add to the dry
components. Rinse the measuring cylinder with a small amount of distilled water and
empty into the vessel. Add distilled water (or the appropriate solvent) to
approximately 90% of the desired volume.
4. Stir the solution using a stir bar and a magnetic stirrer, and adjust the pH with the
appropriate acid or base to the desired value using a calibrated pH meter. Add acid

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orbase drop wise and allow the pH to stabilize before adding further acid or base.
5. Buffer pH changes with temperature. If you will be working with buffers at a specific
temperature, e.g., 4°C, prepare buffers using water at 4°C. Ensure that your pH meter
is adjusted to compensate for the change in temperature.
6. Add distilled water to the desired volume.
7. Filter-sterilize buffer through a 0.45 μm filter, or autoclave.
Solutions that will be used in experiments should not be autoclaved that are sensitive to
bacterial endotoxins and filter-sterilization is recommended in-place of autoclaving.

Storing buffers and solutions

In general, buffers and solutions should be stored at 2–8°C. Solutions containing unstable
compounds (e.g., antibiotics) should be stored in aliquots at –20°C. Bottles should be clearly labelled
with the name of the buffer/solution, the pH, the components and their concentration, and the date of
preparation. When preparing buffers from stock solutions check and, if necessary, adjust the pH
before adjusting the buffer to the final volume.

Study questions:

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