INTRODUCTION

DNA isolation is one of the most basic and essential techniques in the study of DNA. The

extraction of DNA from cells and its purification are of primary importance to the field of

biotechnology and forensics. Extraction and purification of DNA are the first steps in the

analysis and manipulation of DNA that allow scientists to detect genetic disorders, produce

DNA fingerprints of individuals, and even create genetically engineered organisms that can

produce beneficial products such as insulin, antibiotics, and hormones.DNA can be extracted

from many types of cells. The first step is to lyse or break open the cell. This can be done by

grinding a piece of tissue in a blender. After the cells have broken open, a salt solution such

as NaCl and a detergent solution containing the compound SDS (sodium dodecyl sulfate) is

added. These solutions break down and emulsify the fat & proteins that make up a cell

membrane. Finally, ethanol is added because DNA is soluble in water. The alcohol causes

DNA to precipitate, or settle out of the solution, leaving behind all the cellular components

that aren't soluble in alcohol.The DNA can be spooled (wound) on a stirring rod and pulled

from the solution at thispoint.

OBJECTIVE
 To know the proper method of extraction of DNA.

MATERIAL AND APPARATUS

E. Coli culture broth, TE buffer, 25% of SDS, phenol: chloroform, sodium acetate,

isopropanol, ethanol, master mix ready-made of buffer 1x, dNTP, MgCl 2, and Taq

Polymerase, primer forward, primer reverse, sterile dH 2O, DNA template, loading dye, DNA

marker, agarose powder, eppendorf tube, water bath, centrifuge, PCR machine,

comb,electrophoresis chamber and transilluminator.

METHOD

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Extraction Of DNA

500 ml of culture broth was taken into eppendorf tube

The culture was centrifuged at 10,000 rpm for 1 min

Then, 700 l TE buffer was added with 50 l of 25% of SDS and was incubated in
water bath for 1 min at 60oC

300 l of the upper layer was taken into new ependorf tube. Then, 300 l of sodium
acetate and 600 l of isopropanol were added together and mixed gently

Then, it was centrifuged for 1 min at 12,000 rpm

After centrifuged, the supernatant was discarded

500 l of 70% of ethanol was added and centrifuged for 5 min at 12,000 rpm

The solution was discarded and the pallet was dried

Then, 30 l of TE buffer was added

RESULTS

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Figure of the pellet of DNA and detection of DNA band.

Pellet of DNA

Figure 1: The pellet of DNA

DISCUSSION

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The purpose of TE buffer is to protect DNA or RNA from degradation. It is a buffer for
storage of DNA & RNA.

SDS which stands for 'sodium dodecyl sulfate' is a strong anionic detergent that can
solubilize the proteins and lipids that form the membranes. This will help the cell membranes
and nuclear envelopes to break down and expose the chromosomes that contain the DNA.In
addition to removing the membrane barriers, SDS helps release the DNA from histones and
other DNA binding proteins by denaturing them.

The role of isopropanol in DNA extraction is to precipitates the DNA out of solution so it can
be spooled/hooked and re-dissolved in TE buffer.

Ethanol is added in finishing step of DNA extraction as ethanol change ionic potential of
DNA and remove water molecules, which help in precipitation of DNA.

There are some precautions when running the extraction of DNA. DNA extracts contains
irritants so must be handle with care and avoids skin contact. All samples are very sensitive
and should be gently but homogenized thoroughly with the DNA extract reagent.
Homogenization can be achieved by repetitive pipetting with a Pasteur pipette. The sample
will become viscous due to the release of high molecular weight genomic DNA.

The sample cannot be pipette too vigorously as this will shear the genomic DNA. During
washes, DNA can be stored in 95% ethanol for at least 1 week at room temperature or 3
months at 4 C. for long term storage of high molecular DNA, re-precipitate the DNA and
store in ethanol at 4 C. for halting points during isolation, the lysate which contains DNA
extract can be stored for 18 hours at room temperature or 9 months at 4 C or 9 months at -20
C.

Reason why some DNA extraction did not get any band in PCR is there may be due to
personal error while handling both the PCR and DNA extraction methods. It also may be due
to laboratory contamination problem or used of insensitive or inappropriate techniques.
However, the present study and most other studies with negative results have attempted to
optimise methods to ensure optimal sample preparation or inhibition control and they used a
sensitive PCR technique

Another reason for the apparent variability could be that the number determined by the real-
time PCR depends not only on how much DNA is actually present, but also on the amount of

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inhibitors left in the sample, which will add further variation. It could be speculated that
methods with low recovery percentages do not remove inhibitors sufficiently and are prone to
DNA loss during purification, which in combination may give rise to the low recovery as
measured by real-time PCR.

DNA preparation, PCR preparation and PCR were performed in physically separated rooms
as a precaution to avoid contamination.

CONCLUSION

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DNA extraction is very important initial step as if this step is fail then consequently it will
affect the following steps. Therefore, every techniques and surrounding condition need to
take into account in order to minimize the risk of contamination.

REFERENCES

http://bio.lonza.com/uploads/tx_mwaxmarketingmaterial/Lonza_BenchGuides_SourceBook_
Section_I_-_Frequently_Asked_Questions.pdf

http://www.dnr-is.com/src/DNA%20Extract%20Protocol%282%29.pdf

http://www.biomedcentral.com/1471-2180/3/19