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PG & Research Department of Biotechnology& Microbiology,
National College (Autonomous), Tiruchirappalli – 620 001.
1
Distilled water quality- a significant parameter influencing the isolation and
quantification of Deoxyribonucleic Acid
Vasanthakumar P1., Faizsul Raghuman A. N1., Kishore P1., Chinnamani PrasannaKumar1
1PG & Research Department of Biotechnology and Microbiology, National College (Autonomous),
Dindigul road, Trichy- 620001, Tamil Nadu, India
1. Background
As a part of academic practical (Lab in Microbial Genetics and Molecular Biology) requiring the
preparations of high quality DNA, genomic and plasmid DNA isolation was attempted from bacteria
and genomic DNA from fungi. All reagents were prepared from scratch and no commercially
available kits were used. Distilled water for the reagent preparations were generated from the in-
house distillation unit (Borosil). Presented below are the methodologies adopted for DNA
extraction and various troubleshoots performed to overcome the hurdles related to distilled water
quality influencing the isolating high quality DNA.
2. Methodology
2.1. Isolation of bacterial genomic DNA
1. For bacterial isolates from solid media, a loopful of bacterial colony was dispensed into 1 ml of
phosphate buffer saline (PBS) (for 100ml volume: 300 mM NaCl (1.75g), 2.7 mM KCl (0.02g), 10mM
Na2HPO4 (0.14g), 1.7 mM KH2PO4 (0.022g), pH 7.4) in 1.5ml centrifuge tube under clean room
condition.
2. The culture in PBS was well agitated or flash spinned to dispense most of the bacterial cells into the
PBS solution.
3. The inoculation loop was then flame sterilized and kept aside.
4. 1.5ml tube was closed and inverted several times for dispensing the bacterial cells and centrifuged
at 6000 g for 5 minutes.
5. For broth cultures, 1.5ml of culture broth was taken in the 1.5ml centrifuge tube and centrifuged at
6000 g for 5 minutes.
6. The supernatant was carefully discarded and 600 l of lysis buffer (for 500 ml volume: 50mM Tris –
HCl (3.028g), 20mM EDTA (2.922g), 2% SDS (10g), pH 8) was added to the pellet.
PG & Research Department of Biotechnology& Microbiology,
National College (Autonomous), Tiruchirappalli – 620 001.
2
7. The tube was incubated in the water bath at 65C for 1 hour.
8. The mixture was vortexed for 30 seconds every 10 minutes throughout the water bath incubation.
An equal volume (600 l) of Phenol-Chloroform-Isoamyl alcohol (PCI) (25:24:1) reagent was added
and gently mixed by inverting the tube.
9. The tubes were centrifuged at 6000g for 10 minutes and the supernatant was transferred to a fresh
1.5 ml tube.
10. Equal volume of 100% ethanol was added and DNA was precipitated (overnight incubation at -20C
could be followed after ethanol addition for enhanced precipitation).
11. Tubes were centrifuged at 10,000g for 10 minutes and the pellets were air dried (until the ethanol
smell vanishes).
12. 20-50 l (based on the pellet size) of TE buffer (for 100 ml TE buffer: 1M Tris HCl (0.121g), 0.5 M
EDTA (0.029g), pH 8) was added and the DNA was stored at -20C.
2.2. Plasmid DNA isolation
1. Bacteria containing plasmid indicated by its multiple drug resistance pattern in Muller-Hinton agar
(Kirby-Bauer’s method) incubated at 37C overnight was used for plasmid isolation.
2. A loopful of bacterial colony was dispensed into 1 ml of phosphate buffer saline (PBS) (composition
as mentioned in section 2.1.) in 1.5ml centrifuge tube under clean room condition.
3. 1.5ml tube was closed and inverted several times for dispensing the bacterial cells and centrifuged
at 6000 g for 5 minutes.
4. Supernatant was discarded following centrifugation and the bacterial pellet was re-suspended in
100 μl of cold Solution I (Re-suspension buffer: 25 mM Tris-HCl (pH 8), 50 mM glucose, 10 mM
EDTA, stored at 4°C).
5. The tube was vortexed for 2 mins or until all bacteria are fully re-suspended.
6. 200 μl of Solution II (Denaturing solution: 0.2 N NaOH, 1.0% SDS, stored at room temperature) was
added and the tube was carefully inverted 5 times to mix the contents. The contents becoming clear
and thicker was observed as the proteins and DNA are denatured.
7. The tubes were incubated on ice for 5 mins.
8. 150 μl of cold Solution III (Renaturing solution: 120 mL 5M Potassium acetate, 23 mL glacial acetic
acid, 57 mL of dH2O, store Solution III at 4°C) was added to each tube and mixed by inverting
several times. A white precipitate was observed which could contain the bacterial proteins and
genomic DNA.
PG & Research Department of Biotechnology& Microbiology,
National College (Autonomous), Tiruchirappalli – 620 001.
3
9. Tubes were incubated on ice for 5 min. and centrifuged for 5 min. at 12,000 g.
10. The supernatant was collected into a new tube by pipetting or carefully pouring out.
11. 700 μL of cold 100% ethanol or 350 μL room temperature isopropanol was added to the solution to
precipitate the plasmid DNA (or) the tubes were incubated overnight at -20°C or -80°C to improve
precipitation.
12. The tubes were centrifuged at 12,000g for 7 minutes and supernatant was discarded.
13. The pellets were air dried (done by inverting the tube at an angle over kimwipe) for 20-30 minutes
(based on the pellet size). Complete ethanol evaporation could be detect by loss of ethanol smell
from the tube.
14. Pellets were re-suspend with 25-50 μl (based on the pellet size) of TE and stored at -20°C.
2.3. Agarose gel electrophoresis
1. 0.5 g agarose was weighed and dissolved in 50 mL of 1x Tris base Acetic acid
Ethylenediaminetetraacetic acid (TAE) Buffer (100 ml of 50X concentration; Tris Base (24.2g),
Acetic acid (5.71 ml), 0.5M EDTA, pH 8). 250 ml conical flask was used for preparing 50 ml solution
to avoid overflow of gel solution during heating in microwave oven.
2. The solution was heated in microwave oven to boiling constituency (until the solution turns
transparent from translucent).
3. The solution was cooled down to hand bearable temperature and 2μl of Ethidium bromide (EtBr)
solution (10 mg /ml) was added and mixed well by gentle swirling.
4. The solution was then poured in to the gel tray-comb set up (securely tapped gel plates containing
well combs).
5. The solution was allowed to cool to form a gel.
6. The gel was carefully transferred to the electrophoresis tank pre-filled with 1x TAE buffer
(composition as mentioned above).
7. DNA samples were prepared by mixing up to 8μl of DNA sample with 2μl of 5x gel loading dye (5x)
in the insulation tape placed in the floor near the tank.
8. The comb was removed from the gel and the samples were loaded into the well.
9. Appropriate electrodes were connected to the power pack and electropheresis was conducted at
50-100volts for 20min.
10. The movement of the sample was monitored with reference to tracking dye (Bromophenol blue)
and eletrophoresis was terminated when the marker has run 3/4th of the gel's length.
PG & Research Department of Biotechnology& Microbiology,
National College (Autonomous), Tiruchirappalli – 620 001.
4
11. The gel was placed in the UV-trans illuminator and examined for orange colored bands.
PG & Research Department of Biotechnology& Microbiology,
National College (Autonomous), Tiruchirappalli – 620 001.
5
3. Observations
1. The agarose gel and TAE buffer prepared in distilled water had noisier images with filament like
structures with frequent absence of bacterial genomic and plasmid DNA.
2. Results were not improved when freshly prepared 1X TAE buffer was used.
3. Frequently, TAE running buffer produced excess heat which decreased the gel’s rigidity
even at the minimum of 50V power.
PG & Research Department of Biotechnology& Microbiology,
National College (Autonomous), Tiruchirappalli – 620 001.
6
4. When TAE buffer was prepared in commercially available distilled water, the clarity of
the agarose gel was improving and DNA/plasmid could be faintly detected.
5. Further improvement on less noisy gel and DNA detection was witnessed when both
agarose gel and TAE buffer was prepared in commercially available distilled water.
PG & Research Department of Biotechnology& Microbiology,
National College (Autonomous), Tiruchirappalli – 620 001.
7
6. Since the distilled water quality was the issue, we found that when all the reagents for
DNA isolation, preservation, agarose gel electrophoresis was prepared in commercially
available double distilled water, DNA detection was more evident in the agarose gel.
PG & Research Department of Biotechnology& Microbiology,
National College (Autonomous), Tiruchirappalli – 620 001.
8
Fig.: Distillation unit used to produce double distilled water in this study.
Inference
The in-house distilled water exhibited nucleases activity (data not shown) and various
types of invisible debris (evident under UV) which decreased the quality of agarose gel.