MUHIMBILI UNIVERSITY OF HEALTH AND ALLIED SCIENCES

SCHOOL OF MEDICINE
MASTERS OF SCIENCE IN BIOCHEMISTRY

DNA EXTRACTION AND PURIFICATION PRACTICAL REPORT

COURSE :CELL BIOLOGY AND PROTEIN

FACILITATORS : MADAM RUTH AND MR STERWART
STUDENT : ANSELMO MATHIAS MANISHA
DATE OF SUBMISSION: 27/01/20102
 ABSTRACT

In the practical, the extraction of genomic DNA and purification from blood samples was done.
Also, quantity analysis of this DNA was be done by the use of Nano drop spectrophotometer.
DNA extraction is a kind of separation, purification method. I learnt to obtain DNA by help of
lysis and proteases K that destroying structures such as the cell membrane or cell wall. And the
main purpose is observing the DNA extraction by the Mammalian Blood Genomic DNA
Purification Protocol of the Thermo Scientific Gene JET Genomic DNA Purification Kit. The
reason we choose this method is for convenience of manual operating kit available at our genetic
laboratory. Also, we learn measurement of quantity obtained from DNA extraction protocol.
Then we stored the purified DNA for further use in PCR and gel electrophoresis in the coming
practical.

INTRODUCTION TO THE REPORT

Deoxyribonucleic acid (DNA) extraction is the process by which DNA is separated from
proteins, membranes, and other cellular material contained in the cell from which it is recovered.
The several reasons for genomic DNA extraction are:

 Profiling and fingerprinting (blood / sperm sample for comparison and forensic process)
 The first step in the process of gene cloning.
 Characterization and identification (for example transgenic controls modification of
animals).
 To investigate the regulation of gene expression.
 Research genetic disorders or diseases.
 Compatibility testing

Background and literature review

This extraction can be one of the most labor-intensive parts of DNA analysis. Extraction
methods may require an overnight incubation, may be a protocol that can be completed in
minutes or a couple of hours. The DNA extraction process requires careful handling of biological
material to prevent sample contamination and crossover. Tubes should be carefully labeled,
especially when transfers are required.
The extraction of DNA generally follows three basic steps:

 Lyse (break open) the cells.

 Separate the DNA from the other cell components.
 Isolate the DNA.
How to measure absorbance with Scientific Nanodrop Spectrophotometer

Absorbance measurement process is done with spectrophotometer, to measure the concentration

and purity of DNA within the fluid environment that we solve the DNA. We use a UV
spectrophotometer for the determination. Because the DNA by which the solution is the same as
the amount of DNA in the sample absorbed UV amount.

The ratio of 260/280

Nucleic acids at 260 nm, 280 nm for protein gives peaks. Pure DNA example, 260 and 280 nm
absorbance ratio A260nm / A280nm = 1.8.According to this value indicates the efficiency of the
DNA sample in our hands. The value is directly proportional to the proximity of the DNA yield.

Figure 1. Scientific Nanodrop Spectrophotometer Figure 2.example result graph

Principal of Thermo Scientific Gene JET Genomic DNA Purification Kit #K0721
Depending on the starting material, samples are digested with Proteinase K in either the supplied
Digestion or Lysis Solution. RNA is removed by treating the samples with RNase A. The lysate
is then mixed with ethanol and loaded on the purification column where the DNA binds to the
silica membrane. Impurities are effectively removed by washing the column with the prepared
wash buffers. Genomic DNA is then eluted under low ionic strength conditions with the Elution
Buffer.
Materials and reagents as used in DNA purification practical;
Reagents

 Lysis Solution- Used for breaking open cells membrane for use in molecular biology.
 Proteinase K Solution- Digestive enzymes for inactivation of proteins including nucleases.
 Ethanol- Promote ionic bond formations and DNA precipitation
 Wash Buffer I – Often include a low concentration of chaotropic salts to removal protein,
degraded RNA and membrane residue
 Wash Buffer II - An ethanol wash to remove the salts in order to get high yields and
purity.
 Gene JET Genomic DNA Purification Columns pre-assembled with Collection Tubes-
Traps the negatively charged DNA molecules in the silica

Material
 Pipets and pipet tips
 Vortex
 Ethanol (96-100%)
 1.5 mL microcentrifuge tubes
 Microcentrifuge
 Thermomixer, shaking water bath or rocking platform capable of heating up to 56 °C
 Disposable gloves
 Falcon tubes
 Distilled water
Sample
 Human Blood for HLA testing

Objective of the practical

The aim of the practical was to

 Familialize the MSc. Biochemistry student on the principal, procedures and the
manupilation of tools in the genetic laboratory.
 To obtain DNA in a relatively purified form which can be used for further investigation
 METHODOLOGY
After the reception in the laboratory the Facilitator Madam Ruth reminded the students on the
impontant of protective gear, and to adhere to all laboratory rules because we were dealing with
live sample which are Bio-harzadous in nture(Blood).
The following were done in a chronological order as reported below:

Orientation and instrumentation as used in the DNA extraction room of the Genetic laboratory
The students were given a short tour in the laboratory and the following instruments were shown
and description of function were stated;
 Freezer and refrigerator- For storage of reagent and samples used in DNA gel
electrophoresis
 DNA extraction work station- Used for aseptic purification and extraction.
 Vortex machine- Used for mixing and agitation of solution in tubes and flasks.
 Micro centrifuge (16x1000)- Used for centrifugation of solutions
 Hot plate- For heating solutions in test tubes and conical flask
 Heating block- For heating solution in Eppendorf tubes or collection tubes
 Water bath- For boiling or heating reagents
 Automated DNA/RNA/PROTEIN purification system- For biomolecule purification
 Electrophoresis analyzer- Used to read results and display them in Bands on the Screen
 Micro pipette. For accurate measurement of micro little
 Nano drop spectrophotometer- To measure DNA quantity after purification

Decontamination of work station

The work station was cleaned by NaOCl (sodium hypochlorite) throughout, then followed by
95% ethanol, from the areas that were less contaminated to areas with the most prone to
contamination.
This reduces the risk of contamination when cleaning.
Individual equipment’s to be used were also decontaminated by the two solutions starting with
sodium hypochlorite then followed by alcohol.

Calculation of the required amounts of reagents.

Samples to be purified 4 labeled (D88, H88, D89, H89)
Control to the process 1 labeled (N/C)
Minimum error to be expected 10%
Total amount required¿ Total number of samples + expected error
¿ 5 + (5 x 10/100)
 ¿5.5

Total volume = Amount required x Individual volume

Amount required x Individual Volume dispensed
Reagents
volume Required volume in falcon tube
Lysis 400μL x 5.5 2200μL 2.2 ml
Proteinase K 20μL x 5.5 110μL 1.5ml
Ethanol 200μL x 5.5 1100μL 2.8ml
Wash Buffer
I 500μL x 5.5 2750μL 2.8ml
Wash Buffer
II 500μL x 5.5 2750μL 2.8ml
Elution
Buffer 200μL x 5.5 1100μL 1.1ml

Mammalian Blood Genomic DNA Purification Protocol

1. 300μL of whole blood was added in the 5 Eppendorf tubes labeled D88,
H88, D89, H89 and N/C. 400μL of Lysis Solution and 20μL of Proteinase K
Solution were added consecutively to all tubes and mixed thoroughly by
vortexing to obtain a uniform suspension.
2. The samples were incubated at 56 °C for 5 minutes and the vortexed and then
re-incubated for another 5 minutes
3. 200μL of ethanol (96-100%) was added to all 5 samples respectively and
mixed by vertexing
4. The prepared lysates were transferred to a Gene JET Genomic DNA
Purification Columns inserted in a collection tube, then centrifuged for 1 min
at 6000x g.
The collection tubes of each sample with the flow-through solution was
discarded. Gene JET Genomic DNA Purification Column was placed into a
new 2 mL collection tube
5. 500μL of Wash Buffer I (with ethanol added) was added separately to the 5
samples. Centrifuged for 1 min at 8000 x g, then the flow-through were
discarded and the purification columns were placed back into the collection
tube.
6. 500μL of Wash Buffer II (with ethanol added) were added respectively to five
Gene JET Genomic DNA Purification Columns. Then centrifuged for 3 min
at maximum speed (≥12000 x g). Residual solution was seen in the
purification columns, the collection tubes were emptied and the column were
re- spine for 1 min. at maximum speed.
7. The collection tube containing the flow-through solution were discarded and
 the Gene JET Genomic DNA Purification Columns were transferred to a
sterile 1.5 mL Eppendorf tubes
8. 200μL of Elution Buffer were added to the center of the Gene JET Genomic
DNA Purification Columns membrane to elute genomic DNA. then Incubated
for 5 min at room temperature and centrifuge for 1 min at 8000 x g.
9. The purification columns were discarded. Then after the purified DNA was
used from downstream of the Eppendorf tube to measure concentration then
store at -20 °C.
Decontamination of workstation and used equipment’s
The last procedure to be don’t was decontamination of the work station with sodium
hypocrite and absolute alcohol. Various materials used were also decontaminated by
the use of the two solution. The remaining solutions already dispensed and all
unwanted material were discarded in a biohazard waste container.

Results

Measurement of DNA concentration in Nano drop spectrophotometer

Samples Concentration in ng/μl

N/C 0.8
D88 13
H88 4.3
D89 18.3
H89 16.5

Discussion
The protocol used was done with the adjustment of volume of sample which was supposed to be
200μl instead 300μl was used. This may explain the moderate concentration of our purified DNA.
The N/C was supposed to read 0.0 to explain there was no DNA in the sample but instead it
measured 0.8 ng/μl. The results tell us there was a possible contamination with genetic material
or the nano drop spectrometer was not properly cleaned. The required amount needed for a
successive amplification and electrophoresis is 25 ng/μl. From our measurements of D88, D89,
H88, H89 of DNA concentrations of 13, 18.3, 4.3 and 16.5 respectively per a drop of 10μl has to
be recalculated to fit the process. The over all purity and resultant concentration was average
yielding scores not too low, this might be due to pipetting error or spills. From the sample a
comparison with HLA will be between D88 verses H88 and between D89 verses H89 that will be
done in the PCR and gel electrophoresis analysis.
 Conclusion and recommendation on DNA extraction
We were successive in the extraction and purification process; each step was elaborated in a user-
friendly language. The procedures were clearly stated and followed with a little modification to
fit the need of our laboratory. More practice with individual students is necessary to master the
technique of DNA extraction. We are looking forward on the remaining parts of the practical
which includes PCR and gel electrophoresis.

Reference
 Thermo Scientific Gene JET Genomic DNA Purification Kit #K0721,#K0722
 Dahm R (January 2008). "Discovering DNA: Friedrich Miescher and the early years of
nucleic acid research". Human Genetics. 122 (6): 565–81.
 Miller DN, Bryant JE, Madsen EL, Ghiorse WC (November 1999). "Evaluation and
optimization of DNA extraction and purification procedures for soil and sediment
samples"
 Elkins KM (2013). "DNA Extraction". Forensic DNA Biology. pp. 39–52.