MIDLANDS STATE UNIVERSITY

FACULTY OF SCIENCE AND TECHNOLOGY

DEPARTMENT OF APPLIED BIOSCIENCES AND

BIOTECHNOLOGY

NAME: YOLANDA N. MUPITA

REG NO: R217480Y

LEVEL: 2:1

M.O.E: CONVENTIONAL

LECTURER: MS JANI

MODULE: HABB 231

PRAC TITTLE: EXTRACTION OF DNA FROM SPLIT PEAS

ABSTRACT

DNA extraction is a molecular biology technique crucial in research, diagnostics and

biotechnology industry. It allows scientists to isolate DNA molecule from the rest of the cell.
The main objectives of this practical was to isolate plant DNA from split peas . The peas
were firstly softened by adding salt and cold water. Blending was done so as to break the
cells and cell walls. Dish washing liquid was used to break down the cell membrane. Meat
tenderiser was added then followed ethanol. DNA was then observed as fluffy whitish
precipitate.
INTRODUCTION

DNA’s structure was first described by James Watson and Francis Crick in 1953. They used
scientific evidence reported by other scientists to suggest a model for DNA structure as a
double helix. It is composed of nucleotide trisphosphate molecules, referred to as the
‘building blocks’ of DNA. These molecules consist of a trisphosphate group, a deoxyribose
sugar and one of four nitrogenous bases. The four bases involved in a DNA molecule are
adenine and guanine (purines) and thymine and cytosine (pyrimidines). These bases bond to
the deoxyribose sugar and one of the other bases to form base pairs, with adenine and
thymine bonding through two hydrogen bonds, and guanine and cytosine bonding with three
hydrogen bonds. All living organisms contain DNA as it is termed the blueprint of life. DNA
is distinct from cell to cell and this makes it a crucial molecule in cell identification. DNA is
a molecule inside cells which contains the genetic information responsible for the
development and function of an organism. DNA allows this information to be passed from
one generation to the next. DNA is located in the nuclear membrane, chloroplast and
mitochondria in all eukaryotic cells. (Sheehan 2009).

DNA extraction procedures consists of the basic steps of disruption of cell wall, cell
membrane and nuclear membrane to release the DNA into solution, followed by precipitation
of DNA while ensuring removal of the contaminating biomolecules such as proteins,
polysaccharides, lipids, phenols and other secondary metabolites. The process of breaking the
cell is called lysis. This can be done by grinding split peas in a blender. After the cells have
broken open, a salt solution such as NaCl and a detergent solution containing the compound
SDS 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 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 this point
(Wood and Hicks 1983).

The extraction of DNA is important in the field of biotechnology. DNA allows scientists to
detect genetic disorders, produce DNA fingerprints of individuals and even create genetically
organisms that can produce beneficial products such as insulin, antibiotics and hormones.
Hence DNA extraction is important in the fields of forensics where DNA is extracted from
crime scene specimen to find a suspect, used in zoology for breeding to save endangered
species, food industries, pharmaceutical and medicinal industries.
MATERIALS AND METHODS

Materials

Split peas, Salt, Distilled water, dishwashing liquid, Meat tenderizer, Ethanol 96%, Blender,
Strainer, 100ml measuring cylinder, 500ml beaker, test tubes

Method

A pinch of salt and 200ml of cold water were added to 100ml of split peas. They were
allowed to stand overnight to soften. The mixture was blended for 10 minutes and poured
through a strainer into a beaker. 10 ml of dishwashing liquid were then added, mixing was
done and the mixture was then allowed to stand for 10 minutes. 5 ml of mixture were poured
into a test tube. A pinch of meat tenderizer was introduced and stirring was done by gently
inverting the tube. An equal volume of 96% ethanol was finally added and the DNA
appeared.
RESULTS

The DNA appeared fluffy whitish.

Fig 1: DNA precipitates at the bottom of the test tubes

DISCUSSION AND CONCLUSION

To extract DNA, you must remove it from the cells of split peas. Blending the peas with
some salt breaks the cell apart. Salt helps the DNA molecules to stick together while blending
breaks down the cell wall. However, the DNA is still safely contained in the cell because of
the cell and nuclear membrane. Dishwashing liquid which acts as a detergent has polar heads
and non-polar tails. The molecules in detergent are able to pull apart the cell and nuclear
membranes leaving the DNA in the pea soup. (Taylor et al 2004)

The DNA in the pea soup is still covered and protected by the proteins of the cell. The meat
tenderizer which contains enzymes are able to cut apart these proteins to leave the DNA
alone. Ethanol is less dense than water and therefore floats above the pea soup. DNA will not
dissolve in this alcohol so the DNA comes out of the solution or precipitates.

The DNA appeared fluffy white which means it has sheared in the extraction process. The
DNA must have had fine whitish threads which are stringy molecules. The DNA did not
appear the right way because cold ethanol was not used rather room temperature ethanol was
used. Also, ethanol must have been poured on the side of the test tube while its tilted, this
allows to separate soapy cell mixture and ethanol. DNA was extracted too soon without
waiting for 10 minutes for the DNA to actually precipitate. Instead of using dishwasher as a
detergent we should have opted for Cetyltrimethylammonium Bromide (CTAB) to attain
proper results. (Kawaii et al 2003)
REFERENCES

Della porta, S.L. Wood, J. and Hicks, J.B. (1983). A plant DNA mini preparation: Version II.
Plant Molecular Biology Reporter 1: 19-21. Read 13-10-22
Kawai J. and Hayashazaki Y. Philp, Y (2003), DNA book, Cold Spring Harbour laboratory
Press: New York. Retrieved 12-10-22
Sheehan, D. (2009) Physical Biochemistry (2nd ed). United Kingdom: Oxford University
Press.
Taylor, B. Powell, A. John, V (2004). Isolation of Plant DNA and RNA. Focus 4: 4-6.
Retrieved 12-10-22