Isolation

of DAN
DNA isolation is a fundamental technique in
molecular biology that allows scientists to obtain
pure DNA samples for various applications. There
are several methods available for DNA isolation,
each with its advantages and limitations. The
choice of method depends on the sample type, the
intended application, and the resources available.
Proper DNA isolation is crucial for accurate and
reliable downstream analysis, such as PCR, DNA
sequencing, and genetic engineering.
Plasmids are small circular DNA molecules,
most commonly found in bacterial cells (also
found in Cyanobacteria and Fungi). They
replicate in the cells independently of the
chromosomes. The plasmids normally contain
about 2% of total genetic information and
multiply independently of the chromosome.
Requirements for Isolation of Plasmid
DNA
• Tris, Sodium Dodecyl Sulphate (SDS),
tryptone, yeast extract, sodium chloride
(NaCl), agar, potassium- acetate
• Boric acid, glucose, EDTA, sodium
hydroxide.
• Isopropanol, distilled water, high-speed
refrigerated centrifuge, pH meter
micropipettes, watch batch, vortex mixer
• Agarose gel electrophoresis apparatus,
Eppendorf tubes, Tips, glassware
alkaline lysis plasmid isolation
Preparation of Reagents
The following solutions should be prepared in advance:
• Solution I: Add 5 ml of 1M glucose (50mM final concentration of glucose), 0.25
ml of 1M Tris (pH 8.0) (25 mM final concentration of Tris, pH 8.0) and 2 ml of 0.5
M EDTA (pH 8.0) (10mM final concentration of EDTA). By using water make up
the volume to 100 ml (autoclave the solution and store it at room temperature).
• Solution II: Prepare a fresh solution on the day of the experiment by mixing 4 ml
of 5N NaOH (0.2N final concentration of NaOH) and 10 ml of 10% SDS (final
concentration of 1% SDS). By using autoclaved water make the volume of
solution to 100 ml (discard the solution after use).
• Solution III: Prepare a 3M potassium acetate solution of pH 4.8 (autoclave it and
store it at 4°C).
• Running Buffer (for electrophoresis): There are two common types of running
buffers used in agarose gel electrophoresis: Tris-Borate-EDTA (TBE) and Tris-
Acetate-EDTA (TAE).
Procedure of Isolation of Plasmid DNA
• After 24 hours of incubation, take 1.5 ml of culture from the 2 ml culture using an Eppendorf tube
pipette.
• Centrifuge the cells at 6000 rpm for 5-10 minutes. Discard the supernatant completely by inverting
the Eppendorf tube on the blotting paper. Put the Eppendorf tube on ice.
• Completely re-suspend the pellet in (0.1 ml) of ice-cold Solution I to get a uniform suspension. Put
on ice for 5 minutes then keep at room temperature.
• To this suspension, add 0.2 ml of freshly-prepared Solution II. The tube should be closed tightly.
Properly mix the contents by inverting the tube five times.
• Add 0.15 ml of ice-cold Solution III. close the tube tightly and mix the contents properly by
inverting the tube. Keep for 5-7 minutes on ice. Vortex at 10000 to 12000 rpm for 10 minutes at 4°C
• Soon transfer the supernatant to a fresh Eppendorf tube and add 0.45 ml of isopropanol. Gently mix
by inverting the tube and keeping it at room temperature. Take out the supernatant.
• Thereafter, add 0.1 ml of 70% ethanol into the pellet and spin at 10000 rpm for 5 minutes at 4°C.
Then discard the supernatant carefully and dry the tube at 37°C. So that any traces of isopropanol
could be removed.
• Now, add 20 μl of IX-TE from the side and gently tap the tube with your fingers. Add 3.33 μl of 6X
gel loading buffer and run on 1% agarose gel as it has been described below (Note: alternatively, a
plasmid-isolation kit of any company can be used).
Result of Isolation of Plasmid DNA
After precipitating with isopropanol and congratulation, a white precipitate is
observed on the sides or at the bottom of the centrifuge tube. Usually, 2
bands are observed when the plasmid DNA is run on an agarose gel.
These are the supercoiled and relaxed or open circular forms of the
plasmid.
During plasmid isolation no RNase was added, hence a band of RNA will
also be seen on the gel. RNA moves faster than the DNA being small in
size. Therefore, RNA can be distinguished from plasmid DNA.
 Video
https://www.youtube.com/
watch?v=SR4FX6O2u98
 Made by:
Reem
Mamdouh
Romysaa Ehab