This document provides an overview of basic laboratory techniques in molecular biology, emphasizing safety procedures and essential methodologies. Key techniques discussed include DNA isolation, restriction enzyme usage, and the polymerase chain reaction (PCR), which allows for the selective amplification of DNA. The document serves as a guideline for working with DNA and genetic materials in a laboratory setting.

1. Basic Laboratory Techniques
in Molecular Biology
Dr Esther Maina

2. Safety first
Always wear gloves and lab
coat
Never eat or drink in the
lab
Work carefully
If you are not sure – ask
your supervisor

3. Literature
Current protocols in molecular biology
Molecular Cloning: A Laboratory Manual,
Third Edition by Sambrook
www.methods.info

4. Practical components will include:
- Isolation of genomic DNA
- Restriction of DNA
- Ligation into vectors- Plasmids (CLONING)
- Polymerase Chain Reaction (PCR)
-Automated DNA sequencing

5. what is DNA?
1. DNA is deoxyribonucleic acid, a complex string of
syllables found inside your body in tiny genes tightly
packaged in structures called chromosomes
2. The information in your DNA determines your unique
biological characteristics, such as eye color, height,
talents etc.
3. DNA replication is conserved in evolution
4. Molecular biology techniques utilize DNA, RNA, and
enzymes that interact with nucleic acids to understand
biology at a molecular level.

6. Gene
promoter Structural gene
flank flank
upstream downstream
5’ 3’
•A gene is a unit of inheritance
•Carries the information for a:
-polypeptide
-structural RNA molecule

7. Extraction of DNA
Take only DNA from the whole cell extract:
- break cell membrane
- precipitate proteins
Purify DNA from proteins bound, etc

8. DNA Isolation
- Genomic or extra-chromosomal (Plasmid) DNA isolated
-Phenol-chloroform Isoamyl alcohol procedure
-Ethanol precipitation
-Quantified and purity verified by spectrophotometric analysis
-Phenol is naturally somewhat water-soluble, and gives a fuzzy interface,
which is sharpened by the presence of chloroform, and the isoamyl alcohol
reduces foaming
DNA

9. -DNA is precipitated by first ensuring that the
correct concentration of positive ions is present in
solution (too much will result in a lot of salt co-
precipitating with DNA, too little will result in
incomplete DNA recovery)
- 2.5 vols of 95% ethanol is added to the aq
solution in a fresh tube and incubated
-Solution is centrifuged and resulting pellet
washed with 70% ethanol and pellet allowed to dry
-Pellet is subsequently resuspended in either TE
buffer or de-ionised water

10. Nucleases
Endonuclease
5’ Exonuclease 3’ Exonuclease

11. Restriction enzymes
• Specific endonucleases
• Recognize specific short sequences of DNA and cleave
the DNA at or near the recognition sequence
• Recognition sequences: usually 4 or 6 bases but there are
some that are 5, 8, or longer
• Recognition sequences are palindromes
• Palindrome: sequence of DNA that is the same when one
strand is read from left to right or the other strand is
read from right to left– consists of adjacent inverted
repeats

12. Restriction enzymes (cont’d)
• Example of a palindrome:
GAATTC
CTTAAG
• Restriction enzymes are isolated from bacteria
• Derive names from the bacteria
• Genus- first letter capitalized
• Species- second and third letters (small case)
• Additional letters from “strains”
• Roman numeral designates different enzymes from the
same bacterial strain, in numerical order of discovery
• Example: EcoRI
– E Escherichia
– Co coli
– R R strain
– I first enzyme discovered from Escherichia coli R

13. Restriction enzymes play a very important role
-in the construction of recombinant DNA molecules,
as is done in gene cloning experiments
-Another application of restriction enzymes is to
map the locations of restriction sites in DNA

14. Plasmids
Plasmid is an extra-chromosomal DNA molecule separate
from the chromosomal DNA which is capable of
replicating independently of the chromosomal DNA.

15. Safety first
Plasmid is an extra-chromosomal DNA molecule separate
from the chromosomal DNA which is capable of
replicating independently (NON-EPISOMALLY) of
the chromosomal DNA
Genetically
Modified
Organism
Safety information
Biohazard level 1 (S1) – known not pathogenic organism, modification
that does not involve genetic material of higher biohazard level
organisms. Normal lab safety regulation + safety regulation for S1
GMO.

16. Essential plasmids elements
 Origin of replication
 Antibiotic resistance gene (Amp, Kan, Tet, Chl)
 Multiple cloning site

17. Gel electrophoresis analysis of digested DNA
Ethidium bromide stained agarose gel of total RNA (1-
3) and DNA ladder (M)
Plasmid on an agarose gel

19. The Polymerase Chain
Reaction (PCR)
What is the Polymerase Chain Reaction?
• It’s a means of selectively amplifying a particular
segment of DNA
• The segment may represent a small part of a large
and complex mixture of DNAs:
e.g. a specific exon of a human gene
• It can be thought of as a molecular photocopier ( it
mimicks nature by replicating DNA)

20. The Invention of PCR
Invented by Kary Mullis in
1983
• First published account
appeared in 1985
• Awarded NobelPrize for
Chemistry in 1993

21. What’s in the Reaction?
• Template DNA
• Reaction buffer (Tris, ammonium ions (and/or potassium ions), magnesium
ions, bovine serum albumin)
• Nucleotides (dNTPs)
• Primers
• DNA polymerase (usually Taq)
The Basics of PCR Cycling
30–35 cycles each comprising:
– denaturation (95°C),for 30 sec
– annealing (55–60°C), for 30 sec
– extension (72°C), time depends on product size

22. Thermal Cyclers
•PCR cyclers available from many suppliers
•Many block formats and multi-block systems
•Reactions in tubes or 96-well micro-titre plates

23. Has It Worked?
• Check a sample by gel electrophoresis
• Is the product the size that you expected?
• Is there more than one band?
• Is any band the correct size?
• May need to optimize the reaction conditions

24. Optimization
• Annealing temperature of the primers
• The concentration of Mg2+ in the reaction
• The extension time
• The denaturing and annealing times
• The extension temperature
• The amount of template and polymerase —(“more is less”)
Temp. optimization Mg2+ optimization

25. -Do errors occur and does it matter?
. Yes, if you want to clone the amplified DNA — an individual molecule may
harbour several mutations.
• No, if you want to sequence the amplified
DNA or cut it with restriction enzymes.
• Use a proof-reading thermo-stable enzyme
rather than Taq.
- Can I PCR Amplify RNA? Not directly due to DNA
polymerase requiring a DNA template
- Cloning of PCR Products: