Leo Forster                                                                    2213-018International BaccalaureateExtended...
Leo Forster                                                                       2213-018AbstractIt is in the interests o...
Leo Forster                                                                                                               ...
Leo Forster                                                                                                             22...
Leo Forster                                                                                   2213-018Chapter 1: Introduct...
Leo Forster                                                                      2213-0181.2       Background:This investi...
Leo Forster                                                                      2213-018Of the contaminants which are rem...
Leo Forster                                                                     2213-0181.3    Purification:The substance ...
Leo Forster                                                                                   2213-018        1.3.1 Protei...
Leo Forster                                                                      2213-018           1.3.2 Phenol Chlorofor...
Leo Forster                                                                    2213-0181.4    Aim:The aim of this study is...
Leo Forster                                                                                    2213-0181.5     Theoretical...
Leo Forster                                                                                        2213-018           1.5....
Leo Forster                                                                      2213-018           1.5.3 Absorbance Ratio...
Leo Forster                                                                                        2213-018The following t...
Leo Forster                                                                       2213-018       1.5.4 Gel Electrophoresis...
Leo Forster                                                                     2213-018Chapter 2: Methodology2.1    Hypot...
Leo Forster                                                                  2213-0182.2    Procedures:The overall methodo...
Leo Forster                                                                            2213-018           2.2.1 DNA Extrac...
Leo Forster                                                                                    2213-018         For prepar...
Leo Forster                                                                    2213-018       2.2.2 Purification by means ...
Leo Forster                                                                      2213-018       2.2.3 Purification by mean...
Leo Forster                                                                     2213-018   •   The contents of each tube w...
Leo Forster                                                                                   2213-018         2.2.6 Gel E...
Leo Forster                                                                                    2213-018      Chapter 3: Da...
Leo Forster                                                                      2213-018                 3.1.2 Establishm...
Leo Forster                                                                                                       2213-011...
Leo Forster                                                                     2213-011       3.2.2 Absorbance Ratio vs T...
Leo Forster                                                                            2213-0113.3    260/280nm Absorbance...
Leo Forster                                                                                  2213-011 Figure 6: Raw Data f...
Leo Forster                                                                      2213-0113.4    Qualitative Raw Data      ...
Leo Forster                                                                     2213-011       3.4.2     Gel Electrophores...
Leo Forster                                                                  2213-011Gel Electrophoresis was performed twi...
Leo Forster                                                                          2213-011Chapter 4: Discussion of Data...
Leo Forster                                                                  2213-0113x PCTwo further treatments of Phenol...
Leo Forster                                                                       2213-011       4.1.2    Gel Electrophore...
Leo Forster                                                                        2213-011Chapter 5: Evaluation5.1    Lim...
Leo Forster                                                                        2213-0115.2    Further Investigation   ...
Leo Forster                                                                       2213-011Chapter 6: ConclusionDNA was ext...
Leo Forster                                                                   2213-011Chapter 7: Bibliography7.1    Citati...
Leo Forster                                                                    2213-011[13]   Bioteachnology.com. "The Ana...
Leo Forster                                                                   2213-011       Kubo, Ken. "What is DNA Finge...
Leo Forster                                                                                 2213-011Chapter 8: AppendicesA...
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DNA purification using phenol chloroform and proteinase K

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Research work done by my IB student Leo Forester. Please cite and give proper reference to him on his work if you use this material.

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Transcript of "DNA purification using phenol chloroform and proteinase K"

  1. 1. Leo Forster 2213-018International BaccalaureateExtended 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 WordsLeo Forster2213-018 Page 0 of 42
  2. 2. Leo Forster 2213-018AbstractIt is in the interests of secondary schools and universities to have purified DNA for labuse and experimentation. As pure DNA is expensive and unavailable or impractical, acost-effective and efficient alternative for purifying DNA was investigated.Extraction of DNA from onions was done using a SDS/NaCl-based Lysis solution. Theextracts were filtered and DNA precipitated out using ice-cold 95% ethanol. They werestored at 4°C and then purified using different purification protocols. In one case,Phenol Chloroform was added to the sample and DNA precipitated out of the resultingsupernatant. 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 55°C for an hour.Two variations of this also were carried out: one included only the enzyme (PK), whilethe other added a treatment with Phenol Chloroform (PC+PK). Samples were dissolvedin TE Buffer, and 260/280nm absorbance ratio was determined using a UVSpectrophotometer. Gel electrophoresis was also carried out to verify the quantitativedata.Results indicate that with a 260/280nm absorbance ratio of 1.774, 3x PC was able toproduce the purest DNA; while 1x PC and PC+PK gave ratios of 1.585 and 1.621respectively, and PK alone gave a ratio of 1.216. Purity was interpreted based on theassumption that a ratio of 1.8 indicated pure DNA. A greater deviation from 1.8indicates less-pure DNA. These results were reaffirmed in gel electrophoresis, wherebysingle bands of DNA were observed for 3x and 1x PC while other samples containedextensive smearing with very faint or no bands.In conclusion, it was found that 3x PC is a feasible method of producing pure DNA and aviable substitute for expensive commercially purchased DNA. (299 Words) Page 1 of 42
  3. 3. Leo Forster 2213-018ContentsAbstract............................................................................................................................... 1Contents.............................................................................................................................. 2Chapter 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 ................................................................................... 15Chapter 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 ................................................................................... 23Chapter 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 Page 2 of 42
  4. 4. Leo Forster 2213-018 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...................................................................... 31Chapter 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)............................................................. 35Chapter 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 ............................................................................................... 37Chapter 6: Conclusion ...................................................................................................... 38Chapter 7: Bibliography ................................................................................................... 39 8.1 Citations ............................................................................................................ 39 8.2 References........................................................................................................ 40Chapter 8: Appendices ..................................................................................................... 42 Page 3 of 42
  5. 5. Leo Forster 2213-018Chapter 1: Introduction1.1 Rationale of Study:Finding a purification protocol which will produce highly pure DNA at minimalexpenditure allows secondary schools and universities to give a hands-on experiencewith DNA within the lab. An institute wishing to use pure DNA must purchase purifiedLambda DNA1 from commercial retailers. This is inefficient and impractical because theDNA is expensive and highly concentrated. Secondary institutes which allow theirstudents to experience DNA are rare. The result of this study can be used to replaceLambda 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-classinvestigation into the effects several restriction enzymes on DNA, during which the classhad to share the small amount of available Lambda DNA and several students sat outaltogether. 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 anexperiment at all), the goal of this study is to compare several DNA purificationprotocols, 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 Page 4 of 42
  6. 6. Leo Forster 2213-0181.2 Background:This investigation is designed to provide a cheaper alternative for pure DNA used inresearch, development, and schools. It would allow more students to interact with andlearn 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 usingdifferent purification protocols. Onions were chosen because of their availability andcheapness, and because they are a great source of DNA due to the ease of extractionand the lack of safety risks involved as compared to alternatives2.The emphasis of this investigation lies with the further purification of DNA. Thispurification involves removing proteins and other contaminants from the DNA in theonion cells. Figure 1: Allium cepa, the common onion2 DNA extractions from wheat germ and lambda phage are also commonplace Page 5 of 42
  7. 7. Leo Forster 2213-018Of the contaminants which are removed through DNA purification, the most commonare the histone proteins (See Figure 2). The histones serve to provide structure to theDNA and allow it to supercoil3. Figure 2: Histones within a strand of DNAOther impurities include RNA and cell matter as well as enzymes and proteins such asDNase4, an enzyme which would otherwise catalyze the hydrolytic cleavage5 ofphosphodiester bonds6 in the DNA backbone.3 When helical strands of DNA coil around themselves to conserve space4 Deoxyribonuclease, and enzyme which will digest and break down DNA strands5 A chemical reaction resulting in the opening of the DNA double helix6 The link between the 3’ and 5’ ends of phosphate groups in DNA molecules Page 6 of 42
  8. 8. Leo Forster 2213-0181.3 Purification:The substance of this study is to compare several different purification protocols andfind one which best balances cost and effectiveness.Effectiveness will be determined with a UV Spectrophotometer (See 1.5.3, AbsorbanceRatio). 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 isknown as the 260/280 Absorbance Ratio.The protocols being evaluated are: Page 7 of 42
  9. 9. Leo Forster 2213-018 1.3.1 Proteinase K Incubation (PK)The enzyme, Proteinase K, is used to digest nucleic acid proteins and removecontaminants from DNA. It was discovered in 1974 in Engyodontium album7. Though it isvery effective in its applications, it is expensive. In this investigation, it is used as acomparison to judge the effectiveness of Phenol Chloroform rather than as a primarypurification protocol.The enzyme is extracted and is stored in powder form until activated in the presence ofa buffer. Upon being activated, it can simply be added to the nucleic acid extract andincubated with the sample for it to function. Proteinase K is functional in temperaturesranging from 0-65°C, and pH ranging from 4-12 [3].The enzyme becomes activated in the presence of Ca2+ ions8. This is an issue in nucleicacids prepared using EDTA9, as EDTA will attack Ca2+ ions. The enzyme is not significantlyinhibited in the presence of EDTA as EDTA will also weakening protein structures [4]. Pros Cons Versatile Degrades / can be inhibited Expensive ($81.5 per 50µl) Harmless Contaminates samples, lowers purity Table 1: Pros and cons of using PK in purification7 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 Page 8 of 42
  10. 10. Leo Forster 2213-018 1.3.2 Phenol Chloroform Suspension (PC)Phenol Chloroform is cheap to obtain and effective in its application. It provides analternative to conventional purification methods. It is added to DNA and after a shortincubation, is centrifuged, and the DNA precipitated out of solution.The compound removes proteins from the nucleic acid through interactions betweenthe Phenol and Water which cause proteins to undergo a conformational change andexit the aqueous- and enter the organic10 solution [5]. The two layers are partitionedand 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 duringpurification, as a more acidic solution would result in a separation of RNA molecules intophases instead of DNA [6].Here, the effects of doing a single Phenol Chloroform purification compared to doingthree were assessed. It was thought that subsequent purification would remove a largerpercentage of the total proteins within the solution. Pros Cons Cheap ($35 for 200ml) Toxic Simple to use Fast Table 2: Pros and Cons of PC purification10 Phenol, so called because of it’s Carbon-based structure. Page 9 of 42
  11. 11. Leo Forster 2213-0181.4 Aim:The aim of this study is to explore which DNA purification protocols are able to removecontaminants from DNA; hence finding the most practical and effective.The purification protocols used in this study are incubation with the enzyme ProteinaseK, suspension in Phenol Chloroform solution, as well as a mixture of the two (incubationwith 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 thetwo 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 tothe absorption (concentration) of DNA, and will also be used to produce an absorbanceratio to compare the purity of individual samples. Gel electrophoresis of producedsamples will be used to give qualitative data of sample purity. 1.4.1 Objectives of StudyHence, the objectives of this study are as follows: • Investigating Phenol Chloroform and Proteinase K’s 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. Page 10 of 42
  12. 12. Leo Forster 2213-0181.5 Theoretical Basis: 1.5.1 DNA ExtractionExtraction of the DNA is the most important step within this study. If the extract doesnot contain DNA, any attempts at further purification will be invalid.During extraction, onions cells are ruptured using a lysis11 solution. Being composed ofSDS12, NaCl, and EDTA13, the lysis solution functions in that SDS disrupts thehydrophilic14 nature of the cell membrane, effectively breaking it open so that DNA canenter the aqueous solution [7].Hence, DNA strands and onion cell remnants are dissolved in the aqueous solution andcan 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 themembrane and breaking the cell open.13 Ethylenediaminetetraacetic acid, used because it attacks ions which would otherwise degrade DNA14 Water-loving; an important aspect of what holds together the cell membrane. Page 11 of 42
  13. 13. Leo Forster 2213-018 1.5.2 Precipitation of DNARemnants of the onion cell organelles and cytoplasm are present within the aqueoussolution, but will be separated from the DNA through precipitation.Precipitation functions in that the added ethanol makes it much easier for the Na+ (fromNaCl) to interact with the PO4(3-) 15 on the DNA, causing the nucleic acid to become lesspositively charged and hence less hydrophilic - leading it to leave the aqueous solutionand 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 -5°C.Also, during precipitation proceeding Phenol Chloroform purification, 1/10th volume of3M 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. Page 12 of 42
  14. 14. Leo Forster 2213-018 1.5.3 Absorbance Ratio (UV Spectrophotometer)Knowing that DNA absorbs light at 260nm while contaminants absorb light at 280nm, itis possible to, by comparison of the respective absorbance bell curves, assess the ratioof DNA to contaminants within a solution. This is because the more light is absorbed bythe sample, the higher the concentration of nucleic acid or contaminants within thesample. A sample of pure DNA will have a high 260nm absorbance and a low 280nmone. Hence the ratio of absorbances will be proportionally higher.This ratio of absorbances is indicative of DNA concentration, as per the Beer LambertLaw17 where it is possible to relate the light absorbed to the concentration of theabsorbing 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 arethe major contaminating factor in the extract. The 260/280nm incorporates this into itsestimation, so it is optimal when considering DNA purity.17 http://elchem.kaist.ac.kr/vt/chem-ed/spec/beerslaw.htm Page 13 of 42
  15. 15. Leo Forster 2213-018The following table describes what can be expected of the components of a samplebased on the calculated Absorbance Ratio. 260/280 Ratio Sample Consistency 1.3 <50% Contaminants 1.5 50% nucleic acid, 50% contaminants 1.8 100% DNA 2.0 100% RNA / Phenol contamination18 Table 4: Components of a sample at varying Absorbance Ratio [10]It should be noted that the DNA sample may not be pure though its ratio is 1.8. This isbecause other contaminants may not be absorbing at 280nm.This information was applied in the case of this study, and a UV Spectrometer with thecapability to produce a 260/280nm absorbance ratio was used for quantification.18 Phenol absorbs at 270nm, so large amounts of residual phenol can create an artificially high ratio. Page 14 of 42
  16. 16. Leo Forster 2213-018 1.5.4 Gel ElectrophoresisGel electrophoresis is used to provide qualitative data to reinforce the conclusionsdrawn from the quantitative data. It consists of allowing samples to travel through a gelbecause of an electrical current. Due to varying densities and sizes of DNA andcontaminants, they will separate visibly within the gel.When placed under a current the DNA in the gel travels through it. The end-position ofthe 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 adye because it is cheap and effective. The gel is submersed in water and destained so apicture can be taken (See Methodology: 2.2.6, Gel Electrophoresis).(See Appendix 2, for more info). Page 15 of 42
  17. 17. Leo Forster 2213-018Chapter 2: Methodology2.1 Hypothesis:It is hypothesized that the 3x Phenol Chloroform purification will produce the DNA ofhighest purity because through successive Phenol Chlorform purifications proteinswhich had escaped previously will be removed.It is hypothesized that the Proteinase K purification will produce DNA of comparablylower purity because Proteinase K itself is an enzyme (that is, a protein) and will remainin the sample during quantification. Hence, the enzyme itself will be measured as animpurity and will lower the absorbance ratio.It is hypothesized that the mixture of Proteinase K and Phenol Chloroform will be able toproduce DNA of similar purity as the Phenol Chloroform protocol because Proteinase Kwill digest all proteins existing within the extracted DNA, while subsequent PhenolChloroform purifications will remove all remnants of Proteinase K enzymes from thesample. Page 16 of 42
  18. 18. Leo Forster 2213-0182.2 Procedures:The overall methodology of this experiment was as follows: Diagram 1: Overview of extraction & purification process. Page 17 of 42
  19. 19. Leo Forster 2213-018 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 60-65°C 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 Solution’s temperature must not exceed 65°C20 95% denotes a solution of 95 parts Ethanol and 5 parts distilled water. Page 18 of 42
  20. 20. Leo Forster 2213-018 For preparation of DNA for purification: • Using a 1,000μl 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 500μl of TE Buffer23.21 Revolutions Per Minute22 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. Page 19 of 42
  21. 21. Leo Forster 2213-018 2.2.2 Purification by means of Phenol Chloroform (1x and 3x) • 200μl of TE Buffer + DNA was split into two microcentrifuge tubes containing 100μl of solution each. 100μl 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) • 100μl 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) Page 20 of 42
  22. 22. Leo Forster 2213-018 2.2.3 Purification by means of Proteinase K (1x and Combination) • 200μl of TE Buffer + DNA was split into two microcentrifuge tubes containing 100μl of solution each. 50μl of activated Proteinase K enzyme was added to each tube. • Tubes were left in a waterbath at 55°C for one hour. • One of the tubes was refrigerated. (this is 1x PK) • 200μl 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 • 20μl 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 Page 21 of 42
  23. 23. Leo Forster 2213-018 • 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). • 100μl 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 100μl TE buffer and was used to autozero the UV Spectrometer (See Figure 6). • A cell was filled with 100μl 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. Page 22 of 42
  24. 24. Leo Forster 2213-018 2.2.6 Gel Electrophoresis • 0.7% agarose 24 was prepared and a gel was poured. • 30μl of 3x PC was loaded into a well and topped with 2μl 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 225 See Appendix 226 Buffer consisting of Tris base, Boric acid, and EDTA. Keeps the DNA deprotonated and soluble in water27 C16H18N3SCl dissolved in water Page 23 of 42
  25. 25. Leo Forster 2213-018 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 5μl Lambda DNA in 95μl TE Buffer. The results were as follows:TE Buffer / μl Lambda DNA / μl % DNA Absorbance at Absorbance Ratio, Concentration / % 260 nm28 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. Page 24 of 42
  26. 26. Leo Forster 2213-018 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 DNATE Buffer / μl Lambda DNA / μl % DNA Absorbance Ratio, Absorbance Ratio, Concentration / % Day 1 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. Page 25 of 42
  27. 27. Leo Forster 2213-011 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: Standard Calibration Curve for Pure DNA at 260nm 0.4 0.35 y = 0.0666x 0.3 R2 = 0.9964260nm Absorbance 0.25 0.2 0.15 0.1 0.05 0 0 1 2 3 4 5 6 % DNA Concentration of Solution / % `
  28. 28. Leo Forster 2213-011 3.2.2 Absorbance Ratio vs Time & Absorbance Ratio vs DNA Concentration `
  29. 29. Leo Forster 2213-0113.3 260/280nm Absorbance Ratio DataExtraction 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 μl of extract stock in 90 μl 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, respectivelySamples not reflected above were not included because it was found that the obtainedAbsorbance Ratio was either erroneous or immeasurable by the UV Spectrophotometer. Page 28 of 42
  30. 30. Leo Forster 2213-011 Figure 6: Raw Data from UV Spectrophotometer of Absorbance Ratio at 260/280 nmIn the preceding table, the varying dilution factors are the result of inconsistent UVSpectrometer readings. If the readings were erroneous, or if the UV Spectrometer gavean error message (sample too concentrated), the sample was diluted until anappropriate reading was obtained.For further calculation, the above data (3.3) was combined and averages found. Theresults 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 Page 29 of 42
  31. 31. Leo Forster 2213-0113.4 Qualitative Raw Data 3.4.1 Precipitation:Proceeding the second precipitation after Phenol Chloroform purification (SeeMethodology, 2.2.4), the samples were visibly different in their consistencies. It washypothesized that these differences after the second precipitation would relate to thepurity 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 precipitationIt was noted that the precipitated DNA in the 1x PC and PC+PK samples was cloudy andunclear, while the 3x PC sample DNA was very stringy and clear. Page 30 of 42
  32. 32. Leo Forster 2213-011 3.4.2 Gel Electrophoresis ResultsAfter obtaining the Absorbance Ratio for each extract sample, gel electrophoresis wasdone 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, andcan be seen in Figure 9 below: Figure 9: Positive Control for Gel Electrophoresis (zoomed in) Page 31 of 42
  33. 33. Leo Forster 2213-011Gel Electrophoresis was performed twice and the results shown in Figures 10 & 11: DNA Band Smearing (contaminants) Figure 10: Results of Gel Electrophoresis 1 Figure 11: Results of Gel Electrophoresis 2Note that the lanes do not appear in the same order. Page 32 of 42
  34. 34. Leo Forster 2213-011Chapter 4: Discussion of Data4.1 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 dataobtained from the UV Spectrophotometer indicates the following:Unpurified29With a 260/280nm absorbance ratio of 1.489, unpurified DNA was used as negativecontrol30. Using the table on page in 1.5.3, it was determined that the extracted samplesof DNA contained approximately 50% contaminants and 50% DNA. This was reinforcedin that samples of undiluted unpurified DNA were very cloudy and discolored, indicatingcontamination. Hence, the extraction protocol was functional as it gave an acceptableratio of DNA to contaminants. Had the extraction been of lower quality, it may havebeen more difficult to produce pure DNA.1x PcA single treatment with Phenol Chloroform increased the 260/280nm absorbance ratioto 1.585. Hence, a single treatment with PC is not sufficient to remove all contaminantsin a sample, but also shows that phenol-chloroforming is effective. Additionally, duringphenol-chloroforming the supernatant after 1x PC was much larger than after 2x or 3xPC. This may indicate that there were too many contaminants in the sample for thevolume of PC which had been added to handle – that maybe a 1x PC done with anincreased 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 33 of 42
  35. 35. Leo Forster 2213-0113x PCTwo further treatments of Phenol Chloroform resulted in a sample with 260/280nmabsorbance ratio of 1.774. Thus, the sample can be considered almost perfectly pureand is comparable to the commercially purchased Lambda DNA (with260/280nm=1.770). It was also demonstrated that further PC trials will increase samplepurity as contaminants will continue to be removed.PKSurprisingly, Proteinase K treatment resulted in a 260/280nm absorbance ratio of only1.216, indicating a sample composition of almost 100% contaminants. Perhaps toomuch Proteinase K was added to the sample, such that the volume of enzyme dwarfedthe volume of DNA. Hence, the spectrophotometer would measure the enzyme as acontaminant and give an accordingly low absorbance ratio. It was impossible todetermine how much of the contaminants were digested by Proteinase K, as theenzyme inhibited the spectrophotometer’s measurements.PK + PCNot surprisingly, a phenol-chloroforming of a PK sample increased its 260/280nmabsorbance 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 orundigested contaminants being removed. Hence, it was thought that PC was able tobring up the sample purity to a level comparable with 1x PC, and that PC and PK weremore or less even in their purifying capability. Page 34 of 42
  36. 36. Leo Forster 2213-011 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 smearingindicates less pure DNA [11]. Linear DNA will produce a band in the gel: the visibility/sizeof this band indicates the amount of DNA in the sample [12]. Hence, the size of the bandcompared to the amount of smearing gives an indication of the ratio of DNA toimpurities within a sample.In Gel Electrophoresis 1, 3x PC and 1x PC displayed visible bands of DNA with evidenceof limited smearing, while PC+PK and PK displayed no visible band and PC+PK inparticular was one large smear. Thus, it can be concluded that 3x PC and 1x PCcontained DNA as well as some contaminant remnants while PC+PK contained smallamounts of DNA and comparatively large amounts of contaminants.In Gel Electrophoresis 2, there was some error with the loading of 1x PC and so itremained in its well. Conversely, 3x PC showed a visible band of DNA with limitedsmearing while PK also displayed evidence of a band of DNA. PC+PK was a single largesmear of contaminants – as previously.Thus, it is not surprising to see that while all other samples showed a very faint or noband with evidence of a large amount of smearing, 3x PC and 1x PC showed evidence ofa clear band of DNA with limited smearing. As these two samples were also the sampleswith 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 3xPC is both the best and most cost effective method of DNA purification. Page 35 of 42
  37. 37. Leo Forster 2213-011Chapter 5: Evaluation5.1 Limitations and Improvements 5.1.1 Phenol Chloroform ContaminationSmall amounts of phenol in a DNA sample can skew the 260/280 absorbance ratiobecause phenol absorbs at 270nm [13]. Hence, it will raise the 260nm absorbance andlower the 280nm absorbance, and the ratio will go up. Small amounts of DNA with largeamounts of phenol may still give a ratio of 1.8. This was observed in samples which werecontaminated with phenol, as their ratio was ~2.0.The phenol contamination can be estimated using the 260/270nm absorbance ratio ofthe sample. Samples uncontaminated by phenol should have a 260/270nm ratio of 1.2. 5.1.2 Time FrameDue to physical time constraints only two extractions were done. Thus, the conclusionsare not statistically relevant, but give a good general trend for what was beingmeasured. Large amounts of time were spent perfecting the extraction procedure, andso there was not enough time to carry out additional extractions 5.1.3 UV SpectrophotometerDue to the sensitive nature of the UV spectrophotometer it is possible that, instead ofDNA concentration, fluctuations in measurement correspond to varying absorbanceratios. This is unlikely given the consistency between the ratios of similar samplesthough. Page 36 of 42
  38. 38. Leo Forster 2213-0115.2 Further Investigation 5.2.1 SolventDifferent, less toxic, organic solvents should be explored so that students might be ableto carry out purification in the classroom. Phenol Chloroform is too toxic for classroomuse. 5.2.2 230/260 & Other RatiosFor 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 agiven sample: it only accounts for contaminants which absorb at 280nm such as proteinsand enzymes.Other ratios to consider include: 230/260 or 320/260nm [14][15]. The 230/260nm ratiowill show contamination by organic compounds, while the 320nm measurement will tellthe contamination of the quartz cuvette by dust and other factors. 5.2.3 DNA SourceExtraction could be done from sources such as Lamda-phage, broccoli, wheat germ, oryeast to investigate yield differences and optimize the application of the 3x PCpurification across species. Page 37 of 42
  39. 39. Leo Forster 2213-011Chapter 6: ConclusionDNA 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 Chloroformproduces samples of higher purity at a lower cost, and is therefore optimal forpurification of onion DNA – as was hypothesized. Page 38 of 42
  40. 40. Leo Forster 2213-011Chapter 7: Bibliography7.1 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 39 of 42
  41. 41. Leo Forster 2213-011[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. Childrens 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 40 of 42
  42. 42. Leo Forster 2213-011 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 41 of 42
  43. 43. Leo Forster 2213-011Chapter 8: AppendicesAppendix 1Materials for preparation of lysis solution:− 12.5g SDS− 2.2g NaCl− 1.1g Sodium Citrate− 0.07g EDTAAppendix 2The process uses a gel made of agarose31 of varying concentration. High agaroseconcentrations resolve small DNA fragments better, while lower agarose concentrationsresolve larger DNA fragments better. In this investigation, 0.7% agarose was used, as itis 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 combis used to ensure that once hardened, the gel will contain wells into which the DNA canbe loaded. The gel is placed in a buffer, and the nucleic acid sample is mixed with asucrose-based loading dye and inserted into the well. The buffer is placed under acurrent. Due to the negatively charged phosphate backbone of the DNA strands, theywill migrate with the current – through the gel. The rate of their migration depends ontheir size and weight, and so gel electrophoresis is commonly used to separate nucleicacid samples into fragments by size.Methylene Blue works as a dye because it binds to the DNA molecules, and so theyappear darker than the gel background.31 A polysaccharide extracted from algae and seaweed; used because it does not interfere with theproteins and nucleic acids during electrophoresis. Page 42 of 42

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