Practical 4: Isolation of DNA from yeast

Results

Part A

Yeast (5g)

Major contaminants: Grinded with sand

DNA, RNA, proteins, lipids and metabolites
(Anon, 2010).

Sodium chloride and EDTA is used to degrade 20 ml of 0.15 sodium chloride in 0.1M
proteins and RNA while SDS strongly EDTA buffer at pH 8 is added and stirred
destabilize the lipid bilayer of the cell in 2 ml of 10% sodium dodecylsulfate.
membrane (Anon, 2010). (SDS)

Major contaminants:
The broken cell suspension is transferred
Nucleic acid
to a conical flask (200 ml) and stoppered.

Warmed in 60℃. (10 minutes)

The suspension was cooled at room

Major contaminants:
temperature under the tap water.

Nucleic acid, the interphase and lower organic

phase (contains cell debris and denatured
proteins).

Chloroform denatures proteins, avoids the 5 ml of 6M sodium perchlorate and 30

retention of water in the organic phase and ml of chloroform-isoamyl alcohol was
improves phase separation by increasing the added. The suspension is then stirred and
density of the organic phase. Isoamyl alcohol shaken for 15 minutes and centrifuged
aims at RNA isolation and guarantees the for 3 minutes
deactivation of RNases (Green & Sambrook,
2012).

Major contaminants: The upper aqueous phase containing the

DNA and RNA DNA was carefully withdrawn by using
the pipette after being centrifuged

90 ml of ethanol was gently layered over

the aqueous phase. The fibrous
precipitate was collected after mixing
gently with stirring rod

Precipitated DNA was dissolved in 5 ml

Major contaminant:
dilute NaCl-EDTA solution
DNA

The absorbance of the solution at 260nm

and 280nm was measured

Part B
1) Preparation of 1:50 dilution.

Initial Volume = 0.1 mL

Final Volume = 5.0 mL

2) Absorbance at 260 nm, A260 = 0.573

3) Absorbance at 280 nm, A280 = 0.326

Questions

1. What is the concentration of DNA in your purified DNA solution (step 6)?

DNA concentration (original) = 50µg/mL x A260 x dilution factor

= 50µg/mL x A260 x (Final Volume/ Initial Volume)

= 50µg/mL x 0.573 x (5.0/1.0)

= 1432.5 µg/mL

The concentration of purified DNA solution is 1432.5 µg/mL.

2. What is the concentration of protein in the solution?

Protein concentration = 1.45A280 – 0.74A260

= 1.45(0.326) – 0.74(0.573)

= 9.218 x 10-2 mg/mL

The concentration of protein is 9.218 x 10-2 mg/mL.

3. What is the percentage purity of the DNA? Why is it necessary to use quartz cuvettes
in part B?

% purity of DNA = (A260/A280) x 100

= (0.573/0.326) x 100

= 176 %
The percentage purity of DNA is 176 %. To evaluate DNA purity, (A /A ) = (0.573/0.326) =
260 280

1.76. A good-quality DNA usually shows A /A ratio of 1.7-2.0 (Promega Corporation

260 280

2017). Therefore, a ratio of 1.76 is generally accepted as “pure” DNA.

Quartz cuvettes are used in part B for measurement of absorbance of solution at both 260nm
and 280nm instead of plastic and glass cuvettes, this is because plastic and glass cuvettes
absorb most of the light under 280 and 320 nm. This will result the absorbance becomes too
high and affect the reading. Therefore, it is necessary to use quartz cuvette in the case of
shorter wavelengths (260nm and 280nm) as it does not absorb any wavelength from the light
source (Csizmadia n.d.).

Discussion
The technique that we use to determine the concentration of DNA is by measuring the
absorbance of DNA in different wavelength of light. This technique is very common and
easier to carry out as the instrument and material needed are only the spectrophotometer
equipped with UV lamp, UV-transparent cuvettes and a purified DNA solution. DNA will
absorb light most strongly at 260nm (A ). The reading of A260 should be between 0.1-1.0, to
260

ensure that the reading is useful. Based on the result, the absorbance value of A 260 is 0.573,
hence the concentration of DNA is in normal range (Promega Corporation 2012).

However, this absorbance method may have an inaccurate results. Presence of

contaminants in the DNA solution will affect the reading of absorbance. For instance, RNA
molecules and aromatic amino acids have greater absorbance in 260nm and 280 nm. They
will increase the absorbance value if they present in the DNA solution. In addition, there is
higher 260nm absorbance if guanidine presents in the DNA solution. Therefore, as the value
of A260nm is used to calculate the yield or concentration of DNA, the quantity of DNA may be
overestimated. A better method to eliminate the problems in absorbance readings is agarose
gel electrophoresis. Fluorescent DNA-binding dyes can also be used to calculate the yield of
DNA (Promega Corporation 2012).

Two aromatic amino acids which are tyrosine and tryptophan show a larger
absorbance value in 280nm. This method is quick and easier. However, it can be disturbed by
substances that have absorbance at 280nm such as DNA (Csizmadia n.d.). According to
Warburg and Christian, the error is considerable as the solution contains more than 20% of
nucleic acids or any very turbid solution (Center for Cocoa Biotechnology Research and
Development n.d.). Hence, the protein concentration of the experiment may overestimated as
extra nuclei acids contribute a higher A280nm.

Conclusion
In conclusion, the yeast cells are disturbed by grinding with sand and then the DNA is
extracted with sodium perchlorate. DNA are precipitated by adding ethanol while the proteins
are precipitated by shaking with chloroform-isoamyl alcohol. The steps of isolation of DNA
and estimation of the purity of the DNA preparation are summarised in the results above.

During the process of isolation of DNA from yeast, there are several things need to
concern. Extreme pH, temperature and low ionic strength should be avoided. This is because
the phosphodiester bond, N-glycosyl linkage between deoxyribose sugar and purine bases,
hydrogen bonds that hold 2 strands of DNA and also the double chain scissions are disrupted
under those extreme conditions. The DNA-ase can spilt the phosphodiester bond and we use
sodium dodecyl sulfate and EDTA (ethylenediamine tetraacetic acid) to stop the activity of
this enzyme.

The concentration of purified DNA solution is 1432.5 µg/mL. This concentration is

considered in normal range. The concentration of protein is 9.218 x 10-2 mg/mL and this
value may not accurate due to the contaminants present in the solution. Quartz cuvettes
absorb a shorter wavelength of light and therefore they will not contribute to a higher value of
absorbance which will affect the protein and DNA concentration.
References
Center for Cocoa Biotechnology Research and Development, n.d.. Determination of protein
concentration by UV absorption [Online]. Available from:
http://www.koko.gov.my/CocoaBioTech/Protein%20Quantitation1.html [Accessed 7
August 2017].

Csizmadia, A.M., n.d.. 4.2. The UV-VIS photometer [Online]. Available from:
http://elte.prompt.hu/sites/default/files/tananyagok/IntroductionToPracticalBiochemistry
/ch04s02.html [Accessed 6 August 2017].

Csizmadia, A.M., n.d.. 4.5. Determination of protein concentration [Online]. Available from:
http://elte.prompt.hu/sites/default/files/tananyagok/IntroductionToPracticalBiochemistry
/ch04s05.html [Accessed 7 August 2017].

Anon, 2010. DNA/RNA isolation [Online]. Available from:

https://www.applichem.com/en/products/laboratory-biochemicals/dnarna-isolation/
[Accessed 6 August 2017].

Green, M.R. and Sambrook, J., 2012. Molecular cloning: a laboratory manual, 4th ed. New
York: Cold Spring Harbour Laboratory Press.

Promega Corporation, 2012. Protocols and application guide [Online]. Available from:
https://nld.promega.com/-/media/files/resources/paguide/letter/chap9.pdf?la=en
[Accessed 7 August 2017].

Promega Corporation, 2017. How do I determine the concentration, yield and purity of a
DNA sample [Online]. Available from:
https://worldwide.promega.com/resources/pubhub/enotes/how-do-i-determine-the-
concentration-yield-and-purity-of-a-dna-sample/ [Accessed 6 August 2017].