INTRODUCTION:

A. Preparation of Genomic DNA From Eukaryote Cells (Blood)

Methods :
1. The blood was spun in 1.5 mL microfuge tube at 8000 rpm for 10 minutes.
2. The supernatant was discarded. The pellet was resuspended in 250L extraction buffer
(10mM Tris-Cl PH 8.0, 0.1 M EDTA, 20 g/mL RNAse A and 0.5% SDS).
3. The ___ was incubated at 37C for 30 minutes.
4. 5 L of Proteinase K (final concentration 200 g/mL) was added and the tube was
swirled nicely and the suspension was incubated in 50C water bath for 1 hour.
5. The suspension was let cooled down at room temperature for 5 minutes.
6. Equal volume of Saturated-Phenol was added followed by soft vortex for 2 minutes. This
step was done in the fume hood.
7. The suspension was spun at 5000 rpm for 5 minutes.
8. In the fume hood, the upper phase of the suspension was removed to a new sterile tube.
Step 6 and 7 was repeated.
9. The upper phase of the suspension was removed to a new sterile tube and an equal
volume of Chloroform was added followed by soft vortex for 2 minutes.
10. The suspension was spun at 5000 rpm for 5 minutes.
11. The upper phase of the suspension obtained was removed to a new sterile tube carried out
in the fume hood. Step 9 and 10 was repeated.
12. In the end, the upper phase (DNA) was removed into a new sterile microfuge tube.
13. 0.1x volume 3M Sodium Acetate and 2x volume 100% Ethanol (chilled) was added
followed by the gently swirling for 4-5 minutes at room temperature.
14. The suspension was incubated at -70C for 10 minutes.
15. The suspension was spun at 12 000 rpm for 10 minutes. The supernatant was discarded
and the pellet was washed with75% Ethanol.
16. The DNA was spun at 12 000 rpm for 10 minutes. The supernatant was removed and the
pellet was dried at room temperature for 5 minutes.
17. The DNA was dissolved in 50L sterile TE Buffer ( 10mM Tris-CL & 1 mM EDTA ).
18. The DNA was analyzed on 0.7% agarose gel electrophoresis.
B. Extraction of DNA genomic from plant sample
Methods:
1. The plant material was grinded with liquid nitrogen in a mortar. The mortar and pestle
were used to crush hard tissue.
2. The ground tissue was transferred to a eppendorf tube

3. 0.5 ml extraction buffer (1 M Tris-HCl pH 8.0, 0.5M EDTA, 1.4 M NaCl, 0.2% 2mercaptoethanol and 2% Hexadecyltrimethyl ammonium bromide) was added and mixed
well.
4. 130 L 10% SDS, 10 L Proteinase K was added and the tube was shook a few times and
incubated at 65C for 15 minutes.
5. The suspension cooled down at room temperature for 5 minutes.
6. Equal volume of Saturated-Phenol was added followed by soft vortex for 2 minutes. This
step was done in the fume hood.
7. The upper phase of suspension was removed to a new sterile tube in the fume hood.
8. Equal volume of Chloroform

Introduction:
DNA molecule is an invisibly thin, very long strand. DNA extraction is a routine procedure used
to isolate DNA from the nucleus of cells. Extraction of DNA is often an early step in many
diagnostic processes used to detect bacteria and viruses in the environment as well as diagnosing
disease and genetic disorders. DNA extraction involve Breaking cells open to release the DNA.
Cells in a sample are separated from each other, often by a physical means such as grinding or
vortexing. Separating DNA from proteins and other cellular debris.
Genomic DNA constitutes the total genetic information of an organism. Genomic DNA
molecules are generally large, and in most organisms are organized into DNAprotein complexes
called chromosomes. This DNA is usually a circular molecule and is present as multiple copies
within these organelles.
Chloroform is mixed with phenol to increase the efficiency of nucleic acid extractions by
reducing losses of RNA at the interphase. The increased efficiency is due to chloroform's ability
to denature proteins and aid in the removal of lipids, thus improving separation of nucleic acid
into the aqueous phase. Phase separation is also enhanced, which assists in the removal of the
aqueous phase with minimal cross contamination from the organic phase. Often isoamyl alcohol
is added to phenol:chloroform to reduce foaming

Plasmid dna. Bacterial plasmids are closed circular molecules of double-stranded DNA that
range in size from 1 to >200 kb. They are found in a variety of bacterial species, where they
behave as additional genetic units inherited and replicated independently of the bacterial
chromosome.
EDTA which stands for Ethylenediaminetetraacetic acid to our sample tissue. EDTA has four
carboxyl groups ( -COOH). In the alkaline condition of the buffer, EDTA becomes negatively
charged. The EDTA ions then form covalent bonds with the divalent cations and prevent them
from reacting with nucleases. As a result, the enzymes are deactivated and will no longer cause a
threat to the DNA.
DNA extraction buffer is used to maintain the pH of a solution and to stop DNases from
working.
Phenol-chloroform extractions are done when you need to purify DNA from a solution that also
has proteins. The DNA will dissolve in the aqueous layer, and everything else will go into the
non-aqueous layer.
TE is a commonly used buffer solution in molecular biology, especially in procedures involving
DNA or
RNA. "TE" is derived from its components: Tris, a common pH buffer, and EDTA, a molecule
that
chelates cations like Mg2+
.
The purpose of TE buffer is to solubilize DNA or RNA, while protecting it from degradation.