IB EE on DNA purification using phenol chloroform

Leo Forster 2213-018

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International Baccalaureate Extended Essay: Biology

Comparison of Phenol Chloroform, Proteinase K, and a combination of the two in

respect to the purity attained in DNA extracted from onions.

3,995 Words

Leo Forster

2213-018


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Abstract It is in the interests of secondary schools and universities to have purified DNA for lab

use and experimentation. As pure DNA is expensive and unavailable or impractical, a

cost-effective and efficient alternative for purifying DNA was investigated.

Extraction of DNA from onions was done using a SDS/NaCl-based Lysis solution. The

extracts were filtered and DNA precipitated out using ice-cold 95% ethanol. They were

stored at 4C and then purified using different purification protocols. In one case,

Phenol Chloroform was added to the sample and DNA precipitated out of the resulting

supernatant. Two variations of Phenol Chloroform purification were carried out: in one,

the process was repeated once (1x PC); in the other it was repeated thrice (3x PC).

Alternatively, the enzyme Proteinase K was added and incubated at 55C for an hour.

Two variations of this also were carried out: one included only the enzyme (PK), while

the other added a treatment with Phenol Chloroform (PC+PK). Samples were dissolved

in TE Buffer, and 260/280nm absorbance ratio was determined using a UV

Spectrophotometer. Gel electrophoresis was also carried out to verify the quantitative

data.

Results indicate that with a 260/280nm absorbance ratio of 1.774, 3x PC was able to

produce the purest DNA; while 1x PC and PC+PK gave ratios of 1.585 and 1.621

respectively, and PK alone gave a ratio of 1.216. Purity was interpreted based on the

assumption that a ratio of 1.8 indicated pure DNA. A greater deviation from 1.8

indicates less-pure DNA. These results were reaffirmed in gel electrophoresis, whereby

single bands of DNA were observed for 3x and 1x PC while other samples contained

extensive smearing with very faint or no bands.

In conclusion, it was found that 3x PC is a feasible method of producing pure DNA and a

viable substitute for expensive commercially purchased DNA.

(299 Words)


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Contents Abstract............................................................................................................................... 1

Contents.............................................................................................................................. 2

Chapter 1: Introduction...................................................................................................... 4

1.1 Rationale of Study:.............................................................................................. 4

1.2 Background: ........................................................................................................ 5

1.3 Purification:......................................................................................................... 7

1.3.1 Proteinase K Incubation (PK) ...................................................................... 8

1.3.2 Phenol Chloroform Suspension (PC)........................................................... 9

1.4 Aim: ................................................................................................................... 10

1.4.1 Objectives of Study .................................................................................. 10

1.5 Theoretical Basis: .............................................................................................. 11

1.5.1 DNA Extraction.......................................................................................... 11

1.5.2 Precipitation of DNA ................................................................................. 12

1.5.3 Absorbance Ratio (UV Spectrophotometer)............................................. 13

1.5.4 Gel Electrophoresis ................................................................................... 15

Chapter 2: Methodology .................................................................................................. 16

2.1 Hypothesis: ....................................................................................................... 16

2.2 Procedures: ....................................................................................................... 17

2.2.1 DNA Extraction and Precipitation ............................................................. 18

2.2.2 Purification by means of Phenol Chloroform (1x and 3x)......................... 20

2.2.3 Purification by means of Proteinase K (1x and Combination).................. 21

2.2.4 Preparation for measurement of Absorption Ratio ................................. 21

2.3.5 Measurement of Absorption Ratio ........................................................... 22

2.2.6 Gel Electrophoresis ................................................................................... 23

Chapter 3: Data Collection and Presentation.................................................................. 24

3.1 Quantitative Raw Data..................................................................................... 24

3.1.1 Establishment of Absorbance-Concentration Relationship...................... 24

3.1.2 Establishment of DNA Degradation over Time......................................... 25


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3.2 Data Presentation ............................................................................................. 26

3.2.1 Standard Calibration Curve for Pure DNA ................................................ 26

3.2.2 Absorbance Ratio vs Time & Absorbance Ratio vs DNA Concentration... 27

3.3 260/280nm Absorbance Ratio Data ................................................................. 28

3.4 Qualitative Raw Data ........................................................................................ 30

3.4.1 Precipitation:............................................................................................. 30

3.4.2 Gel Electrophoresis Results...................................................................... 31

Chapter 4: Discussion of Data .......................................................................................... 33

4.1 Interpretation of Results................................................................................... 33

4.1.1 260/280nm Absorbance Ratio (Quantitative) ......................................... 33

4.1.2 Gel Electrophoresis (Qualitative) ............................................................. 35

Chapter 5: Evaluation....................................................................................................... 36

5.1 Limitations and Improvements......................................................................... 36

5.1.1 Phenol Chloroform Contamination........................................................... 36

5.1.2 Time Frame .............................................................................................. 36

5.1.3 UV Spectrophotometer............................................................................ 36

5.2 Further Investigation ........................................................................................ 37

5.2.1 Solvent...................................................................................................... 37

5.2.1 230/260 & Other Ratios ........................................................................... 37

5.2.2 DNA Source ............................................................................................... 37

Chapter 6: Conclusion ...................................................................................................... 38

Chapter 7: Bibliography ................................................................................................... 39

8.1 Citations ............................................................................................................ 39

8.2 References........................................................................................................ 40

Chapter 8: Appendices ..................................................................................................... 42


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Chapter 1: Introduction

1.1 Rationale of Study: Finding a purification protocol which will produce highly pure DNA at minimal

expenditure allows secondary schools and universities to give a hands-on experience

with DNA within the lab. An institute wishing to use pure DNA must purchase purified

Lambda DNA1 from commercial retailers. This is inefficient and impractical because the

DNA is expensive and highly concentrated. Secondary institutes which allow their

students to experience DNA are rare. The result of this study can be used to replace

Lambda DNA in the classroom, opting instead to use purified onion DNA.

This lack of affordable and easily produced pure DNA became evident during an in-class

investigation into the effects several restriction enzymes on DNA, during which the class

had to share the small amount of available Lambda DNA and several students sat out

altogether. As this is not optimal and does not enhance the learning of each individual

(and as the class can consider itself lucky to have been able to conduct such an

experiment at all), the goal of this study is to compare several DNA purification

protocols, hoping to find one which optimally balances cost and effectiveness.

1 Double-stranded linear DNA extracted from Lambda phage, a virus which infects E. Coli


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1.2 Background: This investigation is designed to provide a cheaper alternative for pure DNA used in

research, development, and schools. It would allow more students to interact with and

learn about DNA while also saving the institutes a significant amount of money:

commercially available Lamda DNA costs $100 per 0.1mg.

In this study, DNA extracted from Allium cepa, the common onion, will be purified using

different purification protocols. Onions were chosen because of their availability and

cheapness, and because they are a great source of DNA due to the ease of extraction

and the lack of safety risks involved as compared to alternatives2.

The emphasis of this investigation lies with the further purification of DNA. This

purification involves removing proteins and other contaminants from the DNA in the

onion cells.

Figure 1: Allium cepa, the common onion

2 DNA extractions from wheat germ and lambda phage are also commonplace


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Of the contaminants which are removed through DNA purification, the most common

are the histone proteins (See Figure 2). The histones serve to provide structure to the

DNA and allow it to supercoil3.

Figure 2: Histones within a strand of DNA

Other impurities include RNA and cell matter as well as enzymes and proteins such as

DNase4, an enzyme which would otherwise catalyze the hydrolytic cleavage5 of

phosphodiester bonds6 in the DNA backbone.

3 When helical strands of DNA coil around themselves to conserve space

4 Deoxyribonuclease, and enzyme which will digest and break down DNA strands

5 A chemical reaction resulting in the opening of the DNA double helix

6 The link between the 3 and 5 ends of phosphate groups in DNA molecules


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1.3 Purification: The substance of this study is to compare several different purification protocols and

find one which best balances cost and effectiveness.

Effectiveness will be determined with a UV Spectrophotometer (See 1.5.3, Absorbance

Ratio). DNA absorbs light at 260 nm, while most contaminants absorb light at 280nm [1].

Hence, a ratio of the two absorbances can be used to estimate DNA purity. This ratio is

known as the 260/280 Absorbance Ratio.

The protocols being evaluated are:


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1.3.1 Proteinase K Incubation (PK) The enzyme, Proteinase K, is used to digest nucleic acid proteins and remove

contaminants from DNA. It was discovered in 1974 in Engyodontium album7. Though it is

very effective in its applications, it is expensive. In this investigation, it is used as a

comparison to judge the effectiveness of Phenol Chloroform rather than as a primary

purification protocol.

The enzyme is extracted and is stored in powder form until activated in the presence of

a buffer. Upon being activated, it can simply be added to the nucleic acid extract and

incubated with the sample for it to function. Proteinase K is functional in temperatures

ranging from 0-65C, and pH ranging from 4-12 [3].

The enzyme becomes activated in the presence of Ca2+ ions8. This is an issue in nucleic

acids prepared using EDTA9, as EDTA will attack Ca2+ ions. The enzyme is not significantly

inhibited in the presence of EDTA as EDTA will also weakening protein structures [4].

Pros Cons

Versatile Degrades / can be inhibited

Expensive ($81.5 per 50l)

Harmless

Contaminates samples, lowers purity

Table 1: Pros and cons of using PK in purification

7 Formerly Tritirachium album, a microscopic fungus. [2]

8 The Proteinase K buffer will activate the enzymes. Hence they are stored in powder form until used.

9 Ethylenediaminetetraacetic acid, used because it attacks ions which would otherwise degrade DNA


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1.3.2 Phenol Chloroform Suspension (PC) Phenol Chloroform is cheap to obtain and effective in its application. It provides an

alternative to conventional purification methods. It is added to DNA and after a short

incubation, is centrifuged, and the DNA precipitated out of solution.

The compound removes proteins from the nucleic acid through interactions between

the Phenol and Water which cause proteins to undergo a conformational change and

exit the aqueous- and enter the organic10 solution [5]. The two layers are partitioned

and the aqueous solution can be removed.

A 25:24:1 solution of Phenol-Chloroform-Isoamyl Alcohol solution was used in this study,

though the Isoamyl Alcohol is not required. It is important it be kept at pH 7 during

purification, as a more acidic solution would result in a separation of RNA molecules into

phases instead of DNA [6].

Here, the effects of doing a single Phenol Chloroform purification compared to doing

three were assessed. It was thought that subsequent purification would remove a larger

percentage of the total proteins within the solution.

Pros Cons

Cheap ($35 for 200ml)

Simple to use

Fast

Toxic

Table 2: Pros and Cons of PC purification

10 Phenol, so called because of its Carbon-based structure.


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1.4 Aim:

The aim of this study is to explore which DNA purification protocols are able to remove

contaminants from DNA; hence finding the most practical and effective.

The purification protocols used in this study are incubation with the enzyme Proteinase

K, suspension in Phenol Chloroform solution, as well as a mixture of the two (incubation

with Proteinase K, followed Phenol Chloroform purification).

Hence, the precise research topic involved with this study is:

Comparison of Phenol Chloroform (1x and 3x), Proteinase K, and a combination of the

two in respect to the purity attained in DNA extracted from onions.

A UV Spectrophotometer will be used to quantify purified DNA and provide data as to

the absorption (concentration) of DNA, and will also be used to produce an absorbance

ratio to compare the purity of individual samples. Gel electrophoresis of produced

samples will be used to give qualitative data of sample purity.

1.4.1 Objectives of Study Hence, the objectives of this study are as follows:

Investigating Phenol Chloroform and Proteinase Ks ability to purify DNA extracts.

Assessing purity of sample via data from UV Spectrophotometer

Separation of DNA extract bands using gel electrophoresis

Comparing the above on the criteria of cost-effectiveness and efficiency.


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1.5 Theoretical Basis:

1.5.1 DNA Extraction

Extraction of the DNA is the most important step within this study. If the extract does

not contain DNA, any attempts at further purification will be invalid.

During extraction, onions cells are ruptured using a lysis11 solution. Being composed of

SDS12, NaCl, and EDTA13, the lysis solution functions in that SDS disrupts the

hydrophilic14 nature of the cell membrane, effectively breaking it open so that DNA can

enter the aqueous solution [7].

Hence, DNA strands and onion cell remnants are dissolved in the aqueous solution and

can be precipitated using ethanol or isopropanol.

11 Solution used to destroy cell membranes, allowing DNA to exit the cell. For preparation, see Appendix 1.

12 Sodium Dodecyl Sulphate, used to denature the proteins in the cell membrane; damaging the

membrane and breaking the cell open. 13

Ethylenediaminetetraacetic acid, used because it attacks ions which would otherwise degrade DNA 14

Water-loving; an important aspect of what holds together the cell membrane.


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1.5.2 Precipitation of DNA

Remnants of the onion cell organelles and cytoplasm are present within the aqueous

solution, but will be separated from the DNA through precipitation.

Precipitation functions in that the added ethanol makes it much easier for the Na+ (from

NaCl) to interact with the PO4(3-) 15 on the DNA, causing the nucleic acid to become less

positively charged and hence less hydrophilic - leading it to leave the aqueous solution

and enter the ethanol [8].

It is important that the ethanol used in precipitation be as cold as possible. In this study,

the ethanol used was about -5C.

Also, during precipitation proceeding Phenol Chloroform purification, 1/10th volume of

3M Sodium Acetate16 is added to the aqueous solution.

15 Phosphate groups found on DNA strands.

16 Sodium Acetate, NaAC added because it facilitates the pelleting of DNA after precipitation.


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1.5.3 Absorbance Ratio (UV Spectrophotometer) Knowing that DNA absorbs light at 260nm while contaminants absorb light at 280nm, it

is possible to, by comparison of the respective absorbance bell curves, assess the ratio

of DNA to contaminants within a solution. This is because the more light is absorbed by

the sample, the higher the concentration of nucleic acid or contaminants within the

sample. A sample of pure DNA will have a high 260nm absorbance and a low 280nm

one. Hence the ratio of absorbances will be proportionally higher.

This ratio of absorbances is indicative of DNA concentration, as per the Beer Lambert

Law17 where it is possible to relate the light absorbed to the concentration of the

absorbing molecule.

The following table describes what compounds absorb light at which wavelength.

Wavelength / nm Chief Absorbing Compound

230 Organic or carbohydrate contaminants

260 DNA and RNA

270 Phenol

280 Proteins

Table 3: The chief absorbing compound at varying wavelengths [9]

This justifies use of the 260/280nm Absorbance Ratio due to the fact that proteins are

the major contaminating factor in the extract. The 260/280nm incorporates this into its

estimation, so it is optimal when considering DNA purity.

17 http://elchem.kaist.ac.kr/vt/chem-ed/spec/beerslaw.htm


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The following table describes what can be expected of the components of a sample

based on the calculated Absorbance Ratio.

260/280 Ratio Sample Consistency

1.3


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1.5.4 Gel Electrophoresis

Gel electrophoresis is used to provide qualitative data to reinforce the conclusions

drawn from the quantitative data. It consists of allowing samples to travel through a gel

because of an electrical current. Due to varying densities and sizes of DNA and

contaminants, they will separate visibly within the gel.

When placed under a current the DNA in the gel travels through it. The end-position of

the DNA will vary depending on its size and the concentration of gel used.

After this, the gel is dyed so that DNA bands can be seen. Methylene Blue was used as a

dye because it is cheap and effective. The gel is submersed in water and destained so a

picture can be taken (See Methodology: 2.2.6, Gel Electrophoresis).

(See Appendix 2, for more info).


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Chapter 2: Methodology

2.1 Hypothesis: It is hypothesized that the 3x Phenol Chloroform purification will produce the DNA of

highest purity because through successive Phenol Chlorform purifications proteins

which had escaped previously will be removed.

It is hypothesized that the Proteinase K purification will produce DNA of comparably

lower purity because Proteinase K itself is an enzyme (that is, a protein) and will remain

in the sample during quantification. Hence, the enzyme itself will be measured as an

impurity and will lower the absorbance ratio.

It is hypothesized that the mixture of Proteinase K and Phenol Chloroform will be able to

produce DNA of similar purity as the Phenol Chloroform protocol because Proteinase K

will digest all proteins existing within the extracted DNA, while subsequent Phenol

Chloroform purifications will remove all remnants of Proteinase K enzymes from the

sample.


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2.2 Procedures:

The overall methodology of this experiment was as follows:

Diagram 1: Overview of extraction & purification process.


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2.2.1 DNA Extraction and Precipitation

For DNA extraction from onions:

Onion was sliced into cubes with maximum dimension of 1 x 1 x 1 cm and put in

a beaker.

Sufficient lysis solution to cover the cubes was added.

Beaker was placed on a hot plate kept at 60-65C 19 for 15 minutes, while stirring.

Solution was placed in an ice bath for several minutes, while still stirring.

Solution was filtered into a new beaker using filter paper, and filtered once more

after that.

For precipitation of extracted DNA:

Filtered solution was poured into test tubes, with approximately 20ml of solution

in each 50ml test tube.

Test tubes were tilted to increase the surface area for reaction, and ice cold 95%

ethanol 20 was slowly added.

Test tubes were capped and refrigerated overnight.

19 Solutions temperature must not exceed 65C

20 95% denotes a solution of 95 parts Ethanol and 5 parts distilled water.


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For preparation of DNA for purification:

Using a 1,000l micropipette with the end cut off, the cloudy/stringy interphase

(See Figure 3) which had appeared was extracted and placed into

microcentrifuge tubes.

Figure 3: DNA present within added ethanol

Microcentrifuge tubes were centrifuged for several minutes at highest speed

(10,000+ RPM21).

The supernatant was removed, leaving pellet untouched.

Ice-cold 70% ethanol22 was added to the microcentrifuge tubes. The pellet was

dislodged and agitated and then centrifuged at maximum speed for 2-3 minutes.

Previous two steps were repeated two more times (Washing with 70% ethanol).

Supernatant was removed and replaced with approximately 500l of TE Buffer23.

21 Revolutions Per Minute

22 70% denotes a solution of 70 parts Ethanol and 30 parts distilled water.

23 Buffer consisting of Tris base and EDTA. Protects DNA from degradation while rendering it soluble.


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2.2.2 Purification by means of Phenol Chloroform (1x and 3x) 200l of TE Buffer + DNA was split into two microcentrifuge tubes containing

100l of solution each. 100l of Phenol Chloroform was added to each.

The microcentrifuge tubes were centrifuged for 5 minutes at highest speed.

The aqueous phases (See Figure 4) of the resulting partitioned solutions were

removed and placed into new tubes. Care was taken not to disturb the existing

inter- or organic phases.

Figure 4: Phase Separation after addition of Phenol Chloroform

One tube was placed in the refrigerator. (this is 1x PC)

100l of Phenol Chloroform was added to the other tube and spun in a

centrifuge for five minutes at maximum speed (10,000+ RPM).

Aqueous phase was removed and transferred into a new microcentrifuge tube.

The previous two steps were repeated once more, and the resulting tube was

placed in the refrigerator. (this is 3x PC)


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2.2.3 Purification by means of Proteinase K (1x and Combination) 200l of TE Buffer + DNA was split into two microcentrifuge tubes containing

100l of solution each. 50l of activated Proteinase K enzyme was added to each

tube.

Tubes were left in a waterbath at 55C for one hour.

One of the tubes was refrigerated. (this is 1x PK)

200l Phenol Chloroform was added to the tube and spun in a centrifuge for 5

minutes at maximum speed.

Aqueous phase was removed and transferred into a new microcentrifuge tube.

Tube was refrigerated. (this is 1x PC+PK)

2.2.4 Preparation for measurement of Absorption Ratio

20l of Sodium Acetate was added to each tube of 1x PC, 3x PC, and 1x PC+PK.

Cooled 95% ethanol was added to each tube in a 2:1 ratio of ethanol to solution.

Tubes were mixed by inversion and agitation, and were refrigerated for at least

12 hours.

The precipitated DNA (see Figure 5) was extracted and placed into a new

microcentrifuge tube.

Figure 5: Precipitated DNA after Phenol Chloroform purification


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The contents of each tube were centrifuged at maximum speed for several

minutes, and were washed with 70% ethanol (see above, 2.2.1 page 19).

100l of TE buffer was added to each tube, and tube was refrigerated.

2.3.5 Measurement of Absorption Ratio

Figure 6: UV Spectrometer used, model Optizen 2120 UV

A Cell was filled with 100l TE buffer and was used to autozero the UV

Spectrometer (See Figure 6).

A cell was filled with 100l of 3x PC and the 260/280nm absorbance ratio was

measured using the UV Spectrometer.

The cell was emptied of 3x PC and rinsed with TE buffer repeatedly.

Previous two steps were repeated for all other DNA samples.


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2.2.6 Gel Electrophoresis 0.7% agarose 24 was prepared and a gel was poured.

30l of 3x PC was loaded into a well and topped with 2l loading dye 25.

The previous step was repeated for all other DNA samples.

Gel was placed in electrophoresis set (See Figure 7), was submersed in 1x TBE

buffer26, and was left under current for 2-3 hours.

Figure 7: Electrophoresis set used

Gel was removed from electrophoresis set and stained using Methylene Blue27

Destaining was carried out until bands and streaks were clearly visible.

24 See Appendix 2

25 See Appendix 2

26 Buffer consisting of Tris base, Boric acid, and EDTA. Keeps the DNA deprotonated and soluble in water

27C16H18N3SCl dissolved in water


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Chapter 3: Data Collection and Presentation

3.1 Quantitative Raw Data

3.1.1 Establishment of Absorbance-Concentration Relationship

In order to establish whether the absorbance of DNA samples at varying concentrations

changes, the UV Spectrometer was used to quantify known concentrations of Lambda

DNA. These concentrations of DNA were obtained from a dilution of stock solution of

5l Lambda DNA in 95l TE Buffer. The results were as follows:

TE Buffer / l Lambda DNA / l % DNA

Concentration / %

Absorbance at

260 nm28

Absorbance Ratio,

260/280 nm

95.000 5.000 5.00 0.270 1.786

97.500 2.500 2.50 0.169 1.769

98.750 1.250 1.25 0.094 1.696

99.375 0.625 0.63 0.057 1.731

99.688 0.312 0.31 0.026 1.857

99.844 0.156 0.16 0.006 1.778

Table 5: Establishment of Absorbance-Concentration Relationship

It is possible to establish that though the concentration of DNA at 260nm changes as the

amount of DNA dissolved in the solution changes, it has no significant bearing on the

purity reading of the sample.

It can be concluded that DNA concentration increases, the 260nm Absorbance will

increase; that the relationship is directly proportional. (See 3.2.1, Standard Calibration

Curve for Lambda DNA).

It can also be concluded that the concentration of DNA does not affect its absorbance

ratio. Hence the extracts obtained in this study are sufficient for obtaining a justified

absorbance ratio.

28 Assuming DNA is pure, the concentration at 260 nm should be indicative of the concentration of DNA.


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3.1.2 Establishment of DNA Degradation over Time

In order to establish whether the absorbance of DNA samples changes as time passes

(degradation), the UV Spectrometer was used to quantify samples of Lambda DNA over

a period of two days. The results were as follows:

Preparing Lambda DNA

TE Buffer / l Lambda DNA / l % DNA

Concentration / %

Absorbance Ratio,

Day 1

Absorbance Ratio,

Day 2

95.000 5.000 5.00 1.786 1.753

97.500 2.500 2.50 1.769 1.601

98.750 1.250 1.25 1.696 1.580

99.375 0.625 0.63 1.731 1.667

99.688 0.312 0.31 1.857 1.301(a)

99.844 0.156 0.16 1.778 - (b)

Average 1.770 1.581

Table 6: Establishment of DNA Degradation over Time

(a) Data point was erroneous and was not considered in the calculation of the average.

(b) Data point was not measured.

There was a large difference between the average absorption ratios on day one and two,

and the DNA samples did degrade after being isolated from the stock. It was noted that

this degradation was mostly limited to those samples of minute DNA concentration.

It can be concluded that the extracted DNA does degrade over time. Thus, the

absorbance ratio of extracted samples should be measured immediately after

purification.


`

Standard Calibration Curve for Pure DNA at 260nm

y = 0.0666x

R2 = 0.9964

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0 1 2 3 4 5 6

% DNA Concentration of Solution / %

2

6

0

n

m

A

b

s

o

r

b

a

n

c

e

3.2 Data Presentation

3.2.1 Standard Calibration Curve for Pure DNA

Using the 260nm absorbance readings obtained from the known Lambda DNA concentrations above, a standard calibration curve for

pure DNA was created as follows:


`

3.2.2 Absorbance Ratio vs Time & Absorbance Ratio vs DNA Concentration


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3.3 260/280nm Absorbance Ratio Data Extraction and purification was done on two occasions: Extraction 1 and Extraction 2.

All data was obtained using the UV Spectrometer, model Optizen 2120 UV.

The recorded data was reported in Table 7 below:

Absorbance Ratio, 260/280 nm

Dilution Factor

10 fold(a)

2 fold(b)

Sample Type Extraction 1(c)

3x PC 1.789 - (d)

1x PC 1.636 -

PK 1.270 -

PC + PK 1.519 1.671

Unpure (-) 1.479 -

Extraction 2(e)

3x PC 1.759 -

1x PC 1.533 -

PK 1.177 1.146

PC + PK 1.621 1.671

Unpure (-)(g) 1.499 -

Lambda (+)(g) 1.770(f) -

Table 7: Raw Data collected from UV Spectrophotometer

(a) 10 l of extract stock in 90 l of TE buffer.

(b) 2 fold dilution of the 10-fold described in (a)

(c) Extraction completed September, 2010

(d) Data point was not collected due to some error with Spectrophotometer

(e) Extraction completed October, 2010

(f) Taken from the average of the data presented in 3.1.1

(g) Negative and Positive control, respectively

Samples not reflected above were not included because it was found that the obtained

Absorbance Ratio was either erroneous or immeasurable by the UV Spectrophotometer.


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Figure 6: Raw Data from UV Spectrophotometer of Absorbance Ratio at 260/280 nm

In the preceding table, the varying dilution factors are the result of inconsistent UV

Spectrometer readings. If the readings were erroneous, or if the UV Spectrometer gave

an error message (sample too concentrated), the sample was diluted until an

appropriate reading was obtained.

For further calculation, the above data (3.3) was combined and averages found. The

results can be seen in Table 8 below:

Absorbance Ratio, 260/280nm

Sample Type Extraction 1 Extraction 2 Average(a)

3x PC 1.789 1.759 1.774

1x PC 1.636 1.533 1.585

PK 1.270 1.162(b) 1.216

PC + PK 1.595(c) 1.646(d) 1.621

Unpure (-)(f) 1.479 1.499 1.489

Lambda (+)(f)

- - 1.770(e)

Table 8: Calculated averages for 260/280nm Absorbance Ratio

(a) Average calculated using the values from Extraction 1 & 2

(b) Value calculated by averaging values from Extraction 2

(c) Value calculated by averaging values from Extraction 1

(d) Value calculated by averaging values from Extraction 2

(e) Taken from the average of the data presented in 3.1.1

(f) Negative and Positive control, respectively


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3.4 Qualitative Raw Data

3.4.1 Precipitation:

Proceeding the second precipitation after Phenol Chloroform purification (See

Methodology, 2.2.4), the samples were visibly different in their consistencies. It was

hypothesized that these differences after the second precipitation would relate to the

purity of the sample as measured with the UV Spectrophotometer.

During the second extraction cycle, this phenomenon was observed as follows:

Figure 8: Varying DNA consistencies after second precipitation

It was noted that the precipitated DNA in the 1x PC and PC+PK samples was cloudy and

unclear, while the 3x PC sample DNA was very stringy and clear.


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3.4.2 Gel Electrophoresis Results

After obtaining the Absorbance Ratio for each extract sample, gel electrophoresis was

done to provide qualitative data and ensure that the samples actually contained DNA

(See Methodology, 2.2.6).

The positive control (Lambda DNA) for the Gel Electrophoresis was run separately, and

can be seen in Figure 9 below:

Figure 9: Positive Control for Gel Electrophoresis (zoomed in)


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Gel Electrophoresis was performed twice and the results shown in Figures 10 & 11:

Figure 10: Results of Gel Electrophoresis 1

Figure 11: Results of Gel Electrophoresis 2

Note that the lanes do not appear in the same order.

DNA Band

Smearing

(contaminants)


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Chapter 4: Discussion of Data

4.1 Interpretation of Results

4.1.1 260/280nm Absorbance Ratio (Quantitative) Assuming that a 260/280nm absorption ratio of 1.8 indicates pure DNA, the data

obtained from the UV Spectrophotometer indicates the following:

Unpurified29

With a 260/280nm absorbance ratio of 1.489, unpurified DNA was used as negative

control30. Using the table on page in 1.5.3, it was determined that the extracted samples

of DNA contained approximately 50% contaminants and 50% DNA. This was reinforced

in that samples of undiluted unpurified DNA were very cloudy and discolored, indicating

contamination. Hence, the extraction protocol was functional as it gave an acceptable

ratio of DNA to contaminants. Had the extraction been of lower quality, it may have

been more difficult to produce pure DNA.

1x Pc

A single treatment with Phenol Chloroform increased the 260/280nm absorbance ratio

to 1.585. Hence, a single treatment with PC is not sufficient to remove all contaminants

in a sample, but also shows that phenol-chloroforming is effective. Additionally, during

phenol-chloroforming the supernatant after 1x PC was much larger than after 2x or 3x

PC. This may indicate that there were too many contaminants in the sample for the

volume of PC which had been added to handle that maybe a 1x PC done with an

increased volume of PC would yield better results.

29 DNA taken directly from the onion extract, without any further processing.

30 Indicator for the relative purity of samples.


Page 34 of 42

3x PC

Two further treatments of Phenol Chloroform resulted in a sample with 260/280nm

absorbance ratio of 1.774. Thus, the sample can be considered almost perfectly pure

and is comparable to the commercially purchased Lambda DNA (with

260/280nm=1.770). It was also demonstrated that further PC trials will increase sample

purity as contaminants will continue to be removed.

PK

Surprisingly, Proteinase K treatment resulted in a 260/280nm absorbance ratio of only

1.216, indicating a sample composition of almost 100% contaminants. Perhaps too

much Proteinase K was added to the sample, such that the volume of enzyme dwarfed

the volume of DNA. Hence, the spectrophotometer would measure the enzyme as a

contaminant and give an accordingly low absorbance ratio. It was impossible to

determine how much of the contaminants were digested by Proteinase K, as the

enzyme inhibited the spectrophotometers measurements.

PK + PC

Not surprisingly, a phenol-chloroforming of a PK sample increased its 260/280nm

absorbance ratio to 1.621. This means that PC was successful in removing the enzyme;

but it was impossible to determine how the increase in ratio was due to enzyme or

undigested contaminants being removed. Hence, it was thought that PC was able to

bring up the sample purity to a level comparable with 1x PC, and that PC and PK were

more or less even in their purifying capability.


Page 35 of 42

4.1.2 Gel Electrophoresis (Qualitative) Gel electrophoresis was used to verify the data produced by the UV spectrophotometer.

As smearing during gel electrophoresis signifies contamination, increased smearing

indicates less pure DNA [11]. Linear DNA will produce a band in the gel: the visibility/size

of this band indicates the amount of DNA in the sample [12]. Hence, the size of the band

compared to the amount of smearing gives an indication of the ratio of DNA to

impurities within a sample.

In Gel Electrophoresis 1, 3x PC and 1x PC displayed visible bands of DNA with evidence

of limited smearing, while PC+PK and PK displayed no visible band and PC+PK in

particular was one large smear. Thus, it can be concluded that 3x PC and 1x PC

contained DNA as well as some contaminant remnants while PC+PK contained small

amounts of DNA and comparatively large amounts of contaminants.

In Gel Electrophoresis 2, there was some error with the loading of 1x PC and so it

remained in its well. Conversely, 3x PC showed a visible band of DNA with limited

smearing while PK also displayed evidence of a band of DNA. PC+PK was a single large

smear of contaminants as previously.

Thus, it is not surprising to see that while all other samples showed a very faint or no

band with evidence of a large amount of smearing, 3x PC and 1x PC showed evidence of

a clear band of DNA with limited smearing. As these two samples were also the samples

with the best quantitative results, they were confirmed as being the most pure.

In conclusion, based on collected qualitative and quantitative data, it was found that 3x

PC is both the best and most cost effective method of DNA purification.


Page 36 of 42

Chapter 5: Evaluation

5.1 Limitations and Improvements

5.1.1 Phenol Chloroform Contamination

Small amounts of phenol in a DNA sample can skew the 260/280 absorbance ratio

because phenol absorbs at 270nm [13]. Hence, it will raise the 260nm absorbance and

lower the 280nm absorbance, and the ratio will go up. Small amounts of DNA with large

amounts of phenol may still give a ratio of 1.8. This was observed in samples which were

contaminated with phenol, as their ratio was ~2.0.

The phenol contamination can be estimated using the 260/270nm absorbance ratio of

the sample. Samples uncontaminated by phenol should have a 260/270nm ratio of 1.2.

5.1.2 Time Frame

Due to physical time constraints only two extractions were done. Thus, the conclusions

are not statistically relevant, but give a good general trend for what was being

measured. Large amounts of time were spent perfecting the extraction procedure, and

so there was not enough time to carry out additional extractions

5.1.3 UV Spectrophotometer

Due to the sensitive nature of the UV spectrophotometer it is possible that, instead of

DNA concentration, fluctuations in measurement correspond to varying absorbance

ratios. This is unlikely given the consistency between the ratios of similar samples

though.


Page 37 of 42

5.2 Further Investigation

5.2.1 Solvent

Different, less toxic, organic solvents should be explored so that students might be able

to carry out purification in the classroom. Phenol Chloroform is too toxic for classroom

use.

5.2.2 230/260 & Other Ratios

For a true indication of sample purity, other absorbing factors should be considered.

The 260/280nm Absorbance Ratio does not provide a complete picture of the purity of a

given sample: it only accounts for contaminants which absorb at 280nm such as proteins

and enzymes.

Other ratios to consider include: 230/260 or 320/260nm [14][15]. The 230/260nm ratio

will show contamination by organic compounds, while the 320nm measurement will tell

the contamination of the quartz cuvette by dust and other factors.

5.2.3 DNA Source

Extraction could be done from sources such as Lamda-phage, broccoli, wheat germ, or

yeast to investigate yield differences and optimize the application of the 3x PC

purification across species.


Page 38 of 42

Chapter 6: Conclusion DNA was extracted from onions and purified using Phenol Chloroform and Proteinase K.

Purity was measured with the 260/280 absorbance ratio, using a UV spectrophotometer.

The results were compared and it was conclusively proven that Phenol Chloroform

produces samples of higher purity at a lower cost, and is therefore optimal for

purification of onion DNA as was hypothesized.


Page 39 of 42

Chapter 7: Bibliography 7.1 Citations [1] Oswald, Nick. Determining DNA Concentration & Purity. BitesizeBio. Available

from http://bitesizebio.com/2007/08/22/dna-concentration-purity/. Internet.

[2] "Proteinase K." Promega Corporation. Available from

http://www.promega.com/tbs/9piv302/9piv302.pdf. Internet. 2011.

[3] Mecadi GmbH. "Isolation of Genomic DNA. Mecadi GmbH. Available from

http://www.proteinasek.com/index.php?id=659&L=1. Internet.

[4] Sigma-Aldrich. "Analytical Enzymes, Proteinase K". Sigma-Aldrich. Available from

http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-

explorer/analytical-enzymes/proteinase-k.html. Internet.

[5] Oswald, Nick. "How Phenol Extraction Works." BitesizeBio. Available from

http://bitesizebio.com/2008/02/12/the-basics-how-phenol-extraction-works/.

Internet.

[6] Uregina. "Phenol/Chloroform Extraction and Ethanol Precipitation." Available

from http://uregina.ca/~ngdann/Bioc422/proj1.htm. Internet.

[7] Strauss, William. "Preparation of Genomic DNA from Mammalian Tissue."

Available from

http://www.nshtvn.org/ebook/molbio/Current%20Protocols/CPI/im1002.pdf.

Internet.

[8] Oswald, Nick. "How Ethanol Precipitation of DNA and RNA Works."BitesizeBio.

Available from http://bitesizebio.com/2007/12/04/the-basics-how-ethanol-

precipitation-of-dna-and-rna-works/. Internet.

[9][10] Nucleic Acids Analysis. Wikimedia Foundation. Available from

http://en.wikipedia.org/wiki/Nucleic_acids_analysis. Internet.

[11] Dube, Shanta. " DNA Agarose Gel Electrophoresis". Life Technologies. Available

from http://www.bio.davidson.edu/courses/molbio/tips/trblDNAgel.html.

Internet.

[12] Bowen, Robert. "Preparing and Running Agarose DNA Gels. Available from

http://www.vivo.colostate.edu/hbooks/genetics/biotech/gels/agardna.html

Internet.


Page 40 of 42

[13] Bioteachnology.com. "The Analysis of DNA or RNA using Its Wavelengths: 230

nm, 260 nm, 280 nm." Available from http://bioteachnology.com/dna/analysis-

dna-rna-wavelengths-230-260-280-nm. Internet.

[14] Held, Paul. The Importance of the 240 nm Absorbance Measurement. BioTek.

Available from http://www.biotek.com/resources/docs/PW200240nmAM.pdf.

Internet.

[15] Thermo Fisher. 260/280 and 260/230 Ratios. Thermo Scientific. Available from

http://www.phenogenomics.ca/transgenics/docs/NanoDrop%20Nucleic-Acid-

Purity-Ratios.pdf. Internet.

7.2 References

Anderson, Nadja. "Restriction Enzyme Analysis of DNA." Biotech Project,

University of Arizona. Available from

http://biotech.biology.arizona.edu/labs/DNA_analysis_RE_student.html.

Internet.

Applied Biosystems. Quantitating RNA. Ambion. Available from

http://www.ambion.com/techlib/tn/94/949.html. Internet.

Boujtita, Nadia. Isolating Genomic DNA from Whole Blood. Cole-Parmer.

Available from

http://www.coleparmer.ca/techinfo/techinfo.asp?htmlfile=isolating-genomic-

DNA.htm&id=1108. Internet.

Children's Medical Research Institute. "Kitchen Style DNA Extraction, Restriction

Enzymes and DNA electrophoresis." Jeans for Genes. Available from

www.jeansforgenes.org.au/ArticleDocuments/51/DNAExtraction.pdf. Internet.

Chomczynski, Piotr. "Single-step method of RNA isolation by acid guanidinium

thiocyanate-phenol-chloroform extraction". SciVerse. Available from

http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-4DYTRY2-

3W. Internet.

Edvotek, Inc. "Isolation of DNA from Onions." Edvotek. Available from

www.edvotek.com/pdf/WR2-031.pdf. Internet.

Hays, Lana. "Introduction to DNA Extractions." Access Excellence. Available from

http://www.accessexcellence.org/AE/AEC/CC/DNA_extractions.php. Internet.


Page 41 of 42

Kubo, Ken. "What is DNA Fingerprinting? Case of the Bloody

Micropipettor." Biotech Project, University of Arizona. Available from

http://biotech.biology.arizona.edu/labs/DNA_Fingerprinting_teach.html.

Internet.

Kubo, Ken. "DNA Extraction from Onion." Biotech Project, University of Arizona.

Available from

http://biotech.biology.arizona.edu/labs/DNA_extraction_onion_teach.html.

Internet.

Kuhn, Dwight. "Onion DNA Extraction." Virtual Lab Book. Available from

http://classic.sidwell.edu/us/science/vlb5/Labs/DNA_Extraction_Lab/Onion_and

_E__coli/onion_and_e__coli.html. Internet.

Lawrence Livermore National Laboratory. "Restriction of Lambda DNA." LLNL.

Available from http://education.llnl.gov/bep/science/10/sLamb.html. Internet.

National Centre for Biotechnology Education. " The Lambda Protocol." University

of Reading. Available from

http://www.ncbe.reading.ac.uk/ncbe/protocols/PDF/LambdaSG.pdf. Internet.

University of Wisconsin. "Extraction of DNA from Onion." UWSP. Available from

www.uwsp.edu/chemistry/tzamis/lab/onion_dna_lab.pdf. Internet.


Page 42 of 42

Chapter 8: Appendices

Appendix 1 Materials for preparation of lysis solution:

12.5g SDS

2.2g NaCl

1.1g Sodium Citrate

0.07g EDTA

Appendix 2 The process uses a gel made of agarose31 of varying concentration. High agarose

concentrations resolve small DNA fragments better, while lower agarose concentrations

resolve larger DNA fragments better. In this investigation, 0.7% agarose was used, as it

is sufficiently low to allow the expected large fragments of DNA to travel in it.

The gel is created by pouring liquid agarose into a gel former; and a well-forming comb

is used to ensure that once hardened, the gel will contain wells into which the DNA can

be loaded. The gel is placed in a buffer, and the nucleic acid sample is mixed with a

sucrose-based loading dye and inserted into the well. The buffer is placed under a

current. Due to the negatively charged phosphate backbone of the DNA strands, they

will migrate with the current through the gel. The rate of their migration depends on

their size and weight, and so gel electrophoresis is commonly used to separate nucleic

acid samples into fragments by size.

Methylene Blue works as a dye because it binds to the DNA molecules, and so they

appear darker than the gel background.

31 A polysaccharide extracted from algae and seaweed; used because it does not interfere with the

proteins and nucleic acids during electrophoresis.

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