TECHNIQUES IN ANSWERING BIOCHEMICAL QUESTIONS, WITH SPECIAL REFERENCE
TO NUCLEIC ACIDS
Larissa Assam (SUNY Oswego)
Dr. Dhrubajyoti Chattopadhyay (University of Calcutta)
INTRODUCTION
ABSTRACT
This project was aimed at gaining a mastery in techniques necessary for handling nucleic acids. These
techniques were used to determine the genealogy of bacteria from soil samples obtained from the banks of the
River Ganges in the Sunderbans forest area. The desired DNA sequence was inserted into plasmid DNA and
replicated in E.coli cells. An Agarose gel was run to see if the recombinant DNA was effectively removed from
the host cells, the DNA was amplified by PCR and another gel was done to ensure that the PCR products were of
the same size. The BLAST search based on 16S rRNA gene sequences indicated that the bacteria present in the
soil sample included: Gramella portivictoriae, Gramella echinicola, Bacillus decolorationis and Microbulbifer
donghaiensis.
WORK FLOW: METHODS and TECHNIQUES
1.
Cloning (previously executed)
2. Transformation
Vector:
Host: E.coli cells
Target gene: 16S rRNA
Conditions: Heat shock: 30
Endonucleases
minutes at 4°C → 90 seconds at
37°C→5 minutes at 4 °C, 37°C
overnight incubation
3. Plasmid Isolation
Analysis of deoxyribose nucleic acid (DNA) is essential in determining the genealogy
of an organism. In order to study the nucleic acids, a mastery of the different
techniques for handling DNA is essential because that will allow one to choose a
technique for the required data. These experiments are aimed at obtaining a mastery
of the techniques required for handling nucleic acids.
Measurement
of
Measurement of
Transformation
isolated
DNA
Isolation of
isolated DNA
of
concentrations
recombinan
concentrations
recombinant
using
Nanodrop
t DNA from
using UV
DNA
Spectrophotometry
E.coli cells
Spectrophotometry
DNA
sequencing of
recombinant
DNA gene
(PCR)
Visualization of
PCR products in
agarose gel
electrophoresis
5.Measuring DNA concentrations
b. Nanodrop Spectrophotometry
4. Plasmid Purification
5.Measuring DNA concentrations
a. UV Spectrophotometry measuring concentrations
of recDNA extracted
Concentration (µg/ml)= A260 x Dilution factor x (50 µg/ml/1
7. Polymerase Chain Reactions
a) Standard (Normal PCR)
Denaturation
94 °C
5’
94 °C
30”
Annealing
(30 Cycles)
52 °C
40”
C1= 70 µg/ml
C4= 55 µg/ml
C5= 60 µg/ml
C6= 175 µg/ml
Elongation
72 °C
1’
b) Nanodrop Spectrophotometry
Denaturation
96 °C
5’
REFERENCES
96 °C
10”
Annealing
(30 Cycles)
50 °C
10”
72 °C
7 minutes
Amplification
of
recombinant
DNA gene
(PCR)
6. Visualization of Isolated recDNA in agarose
gel electrophoresis
260/280 ratio: purity of the sample
260/230 ratio: presence of organic solvents
Sample
1
2
A260) 35
6
45
13
9
10
65
14
15
16
17
C (µg/µl)
97.3
17.0
52.2
17.97
191.2
128.5
219.0
20.1
47.9
220.4
31.4
192.8
161.3
260/280 Ratio
1.86
1.70
1.84
1.87
1.90
1.88
1.89
1.68
1.78
1.90
1.92
1.91
1.90
8. Visualization of standard PCR products in agarose
gel electrophoresis
260/230 Ratio
1.95
1.26
1.92
2.17
2.26
2.15
2.25
1.64
1.66
2.25
1.76
2.22
2.21
9. DNA Sequencing
Elongation
60 °C
4’
4 °C
7‘
Visualization of
recDNA isolation in
agarose gel
electrophoresis
BLAST search: 16S rRNA database
Bacteria found:
Microbulifer donghaensis
Bacillus decolorationis
Gramella echinicola
Gramella portivictoriae
CONCLUSION
The 16S rRNA BLAST search
revealed 4 species of
bacteria which are all
aerobic and marine. There
are several species of
bacteria present in the
Sunderbans forest area.
Further work could be done
to study the genomic
properties of this bacteria
and also on indentifying
unknown species.
Lau, S. C., Wong , P.-K., Tsoi, M., Li, X., Plakhotnikova, I., Dobretsov, S., et al. (2005). Gramella portivictoriae sp. nov., a novel ACKNOWLEDGEMENTS
member of the family Flavobacteriaceae isolated from marine sediment. International Journal of Systematic and I will like to thank SUNY Oswego and Global Lab for this opportunity and for
Evolutionary Microbiology, 2497-2500. Journal of Systematic and Evolutionary Microbiology, 2497-2500.
funding. I will also like to thank all the members of Dr. Chattopadhyay’s lab for
their guidance and assistance throughout this project.
Old, R., & Primrose, S. (1994). Principles of Gene Manipulation. Blackwell Science.