BIOMOLECULAR

TECHNIQUES

Total DNA
Extraction and
Quantification
prepared by alif irfan azmi (55217218014)
 CONTENT
- Objective
- Methodology
- Result and Discussion
- Conclusion

Our Agenda - Recommendation

- Reference

for Today
 First

objectives
To isolate genomic DNA from selected
commercially processed food
products using KAPA Kit Extract
Extraction Kit (KAPA Biosystem).

Second
To determine the quality and quantity
of the isolated DNA via agarose gel
electrophoresis and
spectrophotometric method

Third
To prepare isolated DNA to be used as
DNA template for PCR lab session.
 DNA Sample Extraction

The beef sample The sample was

provided has been cut transferred into 1.5ml
and mince to 2mm2 microcentrifuge tube
using cutter and and add all the The sample was put
mortar component needed. on vortex for 2-3
second and then was
centrifuged at 13000
rpm for 1 minute to
pellet debris

The supernatant that

contain DNA was
transferred into a
fresh 1.5ml tube
 DNA Sample Extraction
1% of agarose was
prepared using 40 g Submerged the gel
of agarose powder with 1X TBE buffer
with 40ml of pcr
grade water

The solution was

heated in microwave 2 µl of λ DNA-HindIII
for 1 minute until the marker with 2 µl of
solution diluted. loading dye were
mixed together.
 DNA Sample Extraction
The electrophoresis
The mixture were tank was closed and
loaded into the lane 1 the powerpack was
and 8 of the gel. set at 75 volt for 75
minutes.

5 µl of DNA sample
with 1 µl of 6x loading The gel undergoing
dye were mixed staining into ethidium
together and it were bromide and
loaded from lane 2 destaining in water
until lane 7 of the gel for 10 minutes
respectively.
 Bio-Spectrophotometric
Analysis

3 µl of DNA was
diluted with 897 µl The result was
The sample was run
deionized water and obtained and
with bio-
obtained the value for recorded.
spectrophotometer.
A230, A260 and A280
result and discussion
Agarose Gel Agarose gel Electrophoresis was
performed to observed DNA that was

Electrophoresis extracted from commercial beef

sample.

Analysis The DNA extraction result was

obtained and labelled in the Figure 1.

The result was obtained after the agar

was immersed in 1x TBE buffer and
was put inside the powerpack at 75
volt for 75 minutes.
Figure 1: Agarose gel electrophoresis result lane A and B 2µl loaded Lambda DNA HindIII
marker, 2 and 6 were indicates the result of DNA extracted.
Agarose Gel Smear that could be seen in the DNA
extraction result
Electrophoresis The smear indicates that the DNA was
Analysis already degraded (Rordorf, D. 2010).

There was no DNA band present in

the result because the band is fainting
in the process when the DNA
denatured so DNA smear could be
seen
Agarose Gel Based on the 1% agarose gel
electrophoresis that run for 1 hour
Electrophoresis shows the DNA extraction result on
lane 2 and 6 both shows in smear.
Analysis The size of the DNA could not be
decided.

Based on the result the DNA quality

was low because the sample DNA used
from processed beef
Biospectrophotomer Analysis
Biospectrometer analysis was performed to shows the concentration of the
DNA and the purity of the DNA for 2 sample of DNA extraction.

The ideal DNA purity was obtained based on the biospectrometer analysis
results.

Based on the both sample result the average of DNA concentration and DNA
purity could be calculated
Sample 1
DNA Concentration = 0.092×50×300
= 1380

DNA Purity = 0.092/0.083

= 1.1084
Sample 2
DNA Concentration=0.191×50×300
=2865
DNA Purity=0.191/0.171

=1.117

Average DNA Concentration Average DNA Purity

(1380+2865)/2 (1.1084+1.117)/2

=2122.5 =1.1127
Biospectrophotomer Analysis
DNA Concentration based on as show in the biospectrophotometer which
were 1383.5 and 2870.6

The DNA Concentration calculated based on the value of A_(260 ) were 1380
and 2865

The purpose of determining A_(260 ) and A_280 of DNA extracted was to

measure protein contamination (Biotek, 2010).
Biospectrophotomer Analysis
According to Desjardins, P., and Conklin, D., it stated that pure nucleic acids
typically yield a 260/280 ratio of 1.8 to 2.0 for DNA and RNA, respectively.

Based on the result the average DNA purity obtained was 1.1127 which was not
in the range of 1.8 to 2.0.

There were protein contamination in the DNA extraction result so that’s why
the DNA purity was not follow as stated from Desjardins, P., and Conklin, D.
conclusion
Based on the result, the average DNA purity
obtained was 1.1127

According to the DNA extracted result on 1%

agarose gel electrophoresis the quality of the
DNA was low

Based on the DNA extracted result the DNA was

been able to be used for PCR
recommendation
DNA extraction is one of the most modern of
the biological sciences. Scientists and doctors
use DNA extraction to diagnose many medical
conditions to genetically engineer both plants
and animals.

To get a good result of DNA extraction, the

pipetting techniques and aseptic techniques
must be improved.
reference Rordorf, D. (2010, July 17). Continued Growth of the Impact
Factors of MDPI Open Access Journals. Molecules,15(6), 4450-
4451. doi:10.3390/molecules15064450

Anonymous (2011, August 23). Understanding and measuring

variations in DNA sample quality. Retrieved May 10, 2019, from
https://www.ogt.com/resources/literature/483_understanding_an
d_measuring_variations_in_dna_sample_quality

BioTek, 2010. Tech Resources – Nucleic Acid Purity Assessment

using A260/280 Ratios. Available
at: http://www.biotek.com/resources/articles/nucleic-acid-
purity-a260a280.html [Accessed 12 May 2019]

Desjardins, P., & Conklin, D. (2010, November 22). NanoDrop

microvolume quantitation of nucleic acids. Retrieved May 13,
2019, from
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3346308/