BIO 106: Cell Biology Laboratory

DNA Extraction and Analysis

Name: Ekinsu Gülten

Experiment Date: 27.04.2021
Submission Date:
Section: Bio106.01
Assistant Name: Gökhan Gür
1. Introduction

DNA isolation is an important process in molecular biology. DNA isolation is

extensively used for constructing genomic libraries genotyping, PCR and cloning [1]. DNA
isolation has 4 crucial and basic steps; cell lysis, removal of protein and RNA, the
concentration of DNA, determination of the purity and quantity of the DNA [2]. There are
principal ways to cell lysis such as chemical disruption, mechanical disruption and
enzymatic disruption [2]. The centrifuge is essential for the separation of particles from
liquids [3]. Even very stable emulsion can be separated with high centrifugal forces [3].
Therefore, centrifuge derives crucial benefits for the DNA isolation process. There are two
different kinds of centrifuge: differential centrifuge and density gradient centrifuge. Also,
gradient centrifuge can be divided into two: rate-zonal and isopycnic centrifuge [4]. Most of
the centrifuges have solely set for speed (revolution per minute, RPM) [5]. In addition,
certain procedures need precise centrifuge conditions which are needed to be expressed
in terms of relative centrifugal force (RCF) that expressed in units of gravity [5]. Removal
of protein and RNA is an essential process of DNA isolation. The role of ethanol in DNA
isolation is to precipitate DNA proteins due to the insoluble characteristic of DNA in the
ethanol [6]. In addition, pH stability is a crucial factor in the DNA isolation process [7].
Therefore, during this process buffers must be used to protect the DNA from pH shifts [7].
Different approaches have been used for extracting DNA. In most of these different
approaches, toxic solvents or enzymes are used [8]. Considering enzymes are expensive
and non-toxicity is important in DNA isolation, this process has been carried out with the
salting-out method [8]. In purifying step, alkaline lysis is an effective technique for the
removal of unnecessary molecules [9]. Considering the determination of purity and
quantity of DNA Spectrophotometry is a suitable device. Comprehensive information on
colour combination can be achieved by spectrophotometry [10]. DNA Restriction digestion
is a process whereabout DNA is cut at specific sites [11]. DNA is cut by type II restriction
enzymes in the process. Type II restriction enzymes cut the DNA in specific positions
dependent on the recognition sequences [12]. Agarose gel includes a microscopic network
of pores and because DNA is negatively charged due to the phosphate backbone, when a
voltage applied across the agarose, negatively charged DNA will move to the positive
electrode [13]. Therefore, due to the size differences of DNA fragments, DNA will spread
differently along with the gel matrix [13]. Ethidium Bromide (EtBr) is a dark crystalline
marker that is frequently used for the identification and visualization of nucleic acid bands
in electrophoresis [14]. In addition, when ethidium bromide absorbs UV light it is seen as
reddish-brown colour [14].
2. Aim

The aim of the experiment was to isolate the genomic DNA for genetic analysis with
lysis, centrifuge, precipitation and to examine the purity and the concentration of the
isolated DNA with spectrometer and gel electrophoresis.

3. Materials

●Banana
●Table-top centrifuge
●Nanodrop
●Chilled blender
●Saline Citrate Buffer
●2.6 M NaCl
●Absolute ethanol
●Plasmid mini Isolation kit

●100% EtOH
●plasmid
●BamHI-HindIII restriction enzyme
●10X restriction buffer
●Sterile water
●Agarose powder
●1X TAE buffer
●Gel Electrophoresis tank
●Microwave oven
●Ethidium Bromide
●6X Loading buffer
●1kb DNA ladder
●Power supply
●UV transilluminator

●Nanodrop spectrophotometer

4.Methods
 Genomic DNA Isolation

1. 15g of banana

2. 15g of sliced ba

3. 150ml 2.6M Na

4. Solution was m

5. Solution was tr

6. 2 volumes of ab

7. Precipitated DN

8. Precipitated DN

Plasmid DNA Isolation

1. Centrifuge 4 min 6 ml E. Coli cell culture at 5000g

9. Supernatant is

10. 250 μl resuspe

11. 250 μl lysis buf

12. Gently mixed

13. Incubated 5 mi

14. 350 μl chilled b

acetate, pH 4.2)

15. Gently mixed

16. Incubated for 5

17. Centrifuged at

18. Transferred sup

19. Centrifuged at

20. 700 μl wash bu

21. Centrifuged at

22. 100 μl elution b

23. Centrifuged at
 Restriction Digestion Analysis

1. The followings are mixed in an eppendorf tube for single digest with solely BamHI
5.2 μl Plasmid DNA (7800 bp)
2 μL 10X Fastdigest Buffer
1 μL BamHI
11.8 μL distilled H2O

2. The followings are mixed in an eppendorf tube for double digest with BamHI and
HindII
5.2 μl Plasmid DNA (7800 bp)
2 μL 10X Fastdigest Buffer
1 μL BamHI
1 μL HindIII
10.8 µl distilled H2O
3. Briefly spin to bring the contests to the bottom of tubes
4. Reactions were Incubated at 37oC for 30 min
5. Digested samples and uncut plasmid were run in in an agarose gel (%1)

5.Results

Figure 1: Genomic DNA isolation nanodrop measurement results of banana DNA

The maximum peak point of the graph is 230nm

Concentration is 240.8 ng/ul
Ratio of A 260/a280= 1.49

Figure 2: Plasmid DNA nanodrop measurement results

The maximum peak point is 260 nm.

Concentration 192.8 ng/µl
260/280 = 1.89
 M 1 2 3 4

Figure 4: 1 kb DNA marker (Solis BioDyne)

Figure 3: DNA Agarose gel result (M: marker, 1: Genomic banana DNA , 2.: Uncut
plasmid, 3: Digested plasmid with BamHI, 4: Digested plasmid with BamHI and
HindIII)

Well 1 genomic DNA is observed

Well 2 uncut plasmid’s 2 form is observed

Well 3 Digested plasmid with BamHI is observed

Well 4 Digested plasmid with BamHI and HindIII is observed

Discussion

Nucleic acids firmly absorb 260nm UV light because of the resonance structure of
pyrimidine and purine bases [15]. UV light absorbance of other wavelengths,
calculating through the absorbance ratios at these wavelengths to the UV
absorbance at 260 nm, can be used as a measurement of the purity of the sample
[16]. Toward A26/A280 ratio, polysaccharide contamination can be assed due to the
light absorbance of polysaccharides which is 230nm [16]. Therefore, the maximum
peak point of the banana DNA is 230nm as a result of the polysaccharide
contamination. In addition, fished out precipitated DNA has not waited enough for
ethanol to evaporate and that procedure caused the impurity of genomic DNA
sample. In the process of running isolating plasmid DNA on an agarose gel, four
different bands (nicked, linear, supercoiled and circular, single-stranded) can be
observed [17].In the carried out experiment supercoiled plasmid band was observed.
Agarose gel is the most adequate way to separate varying sizes of DNA fragments
which range from 100bp to 25kb [18]. Since DNA has a homogeneous ratio of
mass/charge, DNA fragments are separated with respect to size in agarose gel that
proportional to its molecular weight [19]. The rate of migration depends on not only
the size of the DNA fragment but also the agarose concentration and the voltage
applied [19]. For genomic DNA %1 concentrated agarose should be used and for the
products that are bigger than 5 kilobases less than %1 concentrated agarose gel
may be used [20]. If %1 agarose gel wanted to be used, then the voltage applied can
be adjusted [19]. Our genomic DNA is longer than 5kb <%1 agarose gel should have
been used for better visualization. Since %1 agarose gel was used in the experiment,
100V voltage applied to agarose gel for half an hour for proper visualization. In the
restriction analysis results, the wanted band observed around it was proved that the
proper plasmid was used in the experiment.
 References

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Appendices

Micrograms needed to be expressed in terms of micro liter.

1. 192.8 ng/µl= 0.001x 192.8= 0.1928 µg/µl (1 ng=0.001 µg) divide 1µg
1/0.1928=5.2 µl (1 ng=0.001 µg)

2. Water required: 20-(5.2+1+2)= 11.8 µl

3. Water required: 20-(5.2+2+1+1)= 10.8 µl