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Gel electrophoresis of lambda DNA using agarose and restriction enzymes
 

Gel electrophoresis of lambda DNA using agarose and restriction enzymes

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Research work done by my IB student Gina Lee. Please cite and give proper reference to her if you use her work and meaterial.

Research work done by my IB student Gina Lee. Please cite and give proper reference to her if you use her work and meaterial.

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    Gel electrophoresis of lambda DNA using agarose and restriction enzymes Gel electrophoresis of lambda DNA using agarose and restriction enzymes Document Transcript

    • Research Question: Can Bacteriological Agar be a suitable substitute for Agarose interms of resolution for Lambda DNA digested with HindIII enzyme and if so, what is the most optimal concentration that can produce comparable results with those obtained from Agarose medium? International Baccalaureate Diploma Programme Extended Essay Biology Yoojin Lee Candidate Number: 002213-067 Word Count: 3995 Page 0 / 32
    • Acknowledgement I would like to thank: My supervisor, Mr. Lawrence Kok, for all his guidance and dedication,Taejon Christian International School, for granting opportunities and great facilities, My parents for encouraging me and supporting my needs, And My fellow EE mates and dear seniors. Page 1 / 32
    • AbstractDNA gel electrophoresis is a process of separating DNA fragments using an electric field anda gel matrix. Negatively charged DNA fragments move to the positive end and the gel matrixis used as a medium for migration. Because of the gel’s pores, the shortest DNA fragmentmoves faster than the longest DNA fragment, separating the fragments according to theirsizes. Agarose is commonly used, because it is extremely refined, containing the minimumamount of ions and impurities. Since Agarose is highly purified, it is comparably expensive.This research is designed to find the optimum alternative agar and its best concentration forcomparable resolution.Lambda DNA was used to perform gel electrophoresis. When choosing the best agar amongthree alternatives, restriction enzyme digested DNA HindIII and ionized markers were used.Bacteriological Agar had the best resolution among the alternatives. Hence modifications onthe concentration were performed with Bacteriological Agar to find the optimal concentrationfor it to produce comparable results to those by Agarose. In this part of the experiment, DNAwas digested using the enzyme directly and the mixture was incubated for more than 24 hoursfor complete enzyme reaction 0.6%, 0.8%, and 1.0% Bacteriological Agars were tested andcompared to 0.8% Agarose. In both processes, the gel matrices were stained using MethyleneBlue. For further experiment, QUIKView DNA stain was used.Results showed that Bacteriological Agar produced the best resolution. Of differentconcentrations, 0.6% Bacteriological Agar produced the most comparable results to those ofAgarose. 0.6% Bacteriological Agar could separate multiple fragments well and theirlocations were similar to those in Agarose. Methylene Blue stained three fragments on both0.6% and 0.8%, while QUIKView DNA stain stained four on 0.6% and three on 0.8%Bacteriological Agar.In conclusion, 0.6% Bacteriological Agar is a suitable substitute for 0.8% Agarose.(word count 300) Page 2 / 32
    • Table of ContentsAcknowledgement .................................................................................................................... 1Abstract ..................................................................................................................................... 21.0 Introduction ........................................................................................................................ 51.1 Background Information ...................................................................................................... 51.2 Rationale of Study................................................................................................................ 62.0 Hypothesis........................................................................................................................... 83.0 Preliminary Investigation on Different Types of Agar ................................................. 94.0 Lambda HindIII DNA Ladder on 0.8% Agarose ......................................................... 115.0 Methodology for Choosing the Agar .............................................................................. 135.1 Modifying and Facilitating the Gel Slabs .......................................................................... 135.2 Methodologies.................................................................................................................... 14 5.2.1 Preparation of Gels for Four Different Agars ............................................................. 14 5.2.2 Preparation of Lambda DNA Samples ....................................................................... 15 5.2.3 Preparation of Loading and Gel Electrophoresis ........................................................ 15 5.2.4 Staining and Documenting Process ............................................................................ 165.3 Data Collection .................................................................................................................. 175.4 Comparison on Bacto and Bacteriological Agar with Different Concentrations .............. 175.5 Data Analysis ..................................................................................................................... 186.0 Methodology for Testing Different Concentrations of Bacteriological Agar, whichwas chosen from the previous experiments ......................................................................... 196.1 Methodologies.................................................................................................................... 19 Page 3 / 32
    • 6.1.1 Preparation of Gels with Different Concentrations .................................................... 19 6.1.2 Preparation of Lambda DNA Sample Digested with HindIII..................................... 196.2 Data Collection .................................................................................................................. 206.3 Data Analysis ..................................................................................................................... 217.0 Further Investigation on Resolving Six Fragments, using a Different Stain ..................... 238.0 Discussion of the Results ................................................................................................. 269.0 Evaluation ......................................................................................................................... 279.1 Limitations and Uncertainties ............................................................................................ 279.2 Ways of Improvements ...................................................................................................... 289.3 Unresolved Questions and Further Research ..................................................................... 2910.0 Conclusion ....................................................................................................................... 3011.0 Appendix .......................................................................................................................... 3112.0 Bibliography .................................................................................................................... 32 Page 4 / 32
    • 1.0 Introduction1.1 Background InformationGel electrophoresis is a biotechnology used to separate DNA fragments according to itslength using agar medium and electric field. Since DNA is negatively charged because ofphosphate group, it moves to the positive pole of the electric field. As the fragments movethrough the pores of agar medium, the shorter fragments move faster than the longerfragments. As a result, this method separates the DNA fragments according to their sizes.In this experiment, Lambda DNA1 is used. It is approximately 48,000 base pairs long.[1]HindIII, a restriction enzyme, is used to digest the Lambda DNA into 8 fragments: at 23130,9416, 6557, 4361, 2322, 2027, 564, 125 bp2.[2] However, according to Figure 1 below, only 7fragments are shown because the amount of 125bp is too little to notice. Figure 1: 1% Agarose gel when HindIII is applied to Lambda DNA.[3]1 Double-stranded linear DNA from virus particle called Lambda phage2 Unit for base pair. Page 5 / 32
    • Figure 2: structure of Agarose[4]Agarose3, whose chemical formula is shown in Figure 2 above, is commonly used for themedium because it is highly purified, containing the minimum amount of ions and impurities.The presence of ions and impurities impede the migration of fragments through the electricfield when submerged in TBE buffer4. Also, it de-stains quickly after stained with MethyleneBlue5. Since Methylene Blue stains all of the negatively charged ions including DNA, gelswith high ion concentration will appear denser in color and will take longer time to de-stain.1.2 Rationale of StudyGel electrophoresis is commonly taught in high school as well as in colleges. However, oftentimes, not all schools promote the experiment, because of its cost, because Agarose is veryexpensive compared to other less-purified agars. The gel electrophoresis chamber itself costsbetween $150 to $300 and Agarose ranges from $125.28 to $360.04 while BacteriologicalAgar costs only around $20.08. Not only that, other materials and equipments such asLambda DNA, restriction enzymes, micropipettes, and others cost quite a lot as well.This practical skill is somewhat a costly experiment for high school students, because3 Linear polysaccharide polymer of agarobiose4 TBE stands for Tris Borate EDTA and it deprotonates DNA, contains necessary ions, and protects nucleicacids.5 A positively charged dye used for staining DNA fragments in the gel Page 6 / 32
    • Agarose, DNA, restriction enzymes, and other reagents are not recyclable. In order to devisea cheaper method, cost-effective agar substitute must be found. Thus, finding alternative, yetviable substitute will be vital to reduce the economical burden of institutions and to givemore students chance to perform such experiment.Moreover, DNA electrophoresis is often used to determine the size of unknown DNAfragment by comparing it with DNA ladder6. This is also used at crime scenes. HindIIIrestriction enzyme ideally produces eight fragments of various lengths. By comparing HindIIIladder with unknown DNA, a possible length of unknown DNA can be interpolated. Sincethey are run with Agarose, a viable substitute will enable such DNA profiling processedeconomically.For this research, Bacteriological, Bacto, and Agar C, which are used for nutrition agar plates,were chosen. Since the purpose of gel electrophoresis is to observe the separation of DNAfragments clearly, comparing the resolution was essential. Moreover, the pace of the DNAfragment migration is inversely proportional to the concentration of the gel. By changing theconcentrations, the substitute for Agarose can be found, which is much cheaper, yet canproduce comparable results.Thus, the research question is Can Bacteriological Agar be a suitable substitute forAgarose in terms of resolution for Lambda DNA digested with HindIII enzyme and if so,what is the most optimum concentration that can produce comparable results withthose obtained from Agarose medium?6 DNA ladder consists of many DNA fragments of different lengths Page 7 / 32
    • 2.0 HypothesisSince Methylene Blue stains all negatively charged particles, the more ions the gel contains,the denser in color it appears during staining and de-staining process. It is essential that thegel contains the least amount of ions, like the controlled Agarose, so that it can facilitate theprocess and produce results with high resolution.When qualitatively observed, the particles of Agarose were very fine and white as seen inFigure 3 below. The monomer of Agarose form well organized pores when made into gels.Bacteriological Agar is also composed of fine particles. Compared to that, Bacto Agarappeared yellower and composed of relatively larger particles, which suggest that it may formirregular pores. Agar C had fine particles, but was heavily colored. Hence, BacteriologicalAgar may be a better substitute. Figure 3: different agar particlesWhen comparing the liquefied Agarose and Bacteriological Agar, Bacteriological Agarseemed more viscose than Agarose. In higher gel concentration, the fragments migrate at arelatively slow pace, so the separation of smaller fragments are conspicuous, whereas inlower gel concentration the fragments move at fast pace and the separation of largerfragments are more visible. The rate of migration is inversely proportional to theconcentration. Moreover, the resolution, or the intensity of fragments, depends on the amountof DNA. Therefore, if the concentration and the amount of DNA are adjusted, the Page 8 / 32
    • Bacteriological Agar can produce similar results to that of the Agarose. Thus, it ishypothesized that Bacteriological Agar can be a suitable substitute for Agarose in terms ofDNA gel electrophoresis use.3.0 Preliminary Investigation on Different Types of AgarA preliminary investigation was done to research the correlations between the conductivityand pH and the resolution. All of the agars were tested using the Logger Pro7 conductivityprobe and pH sensor.Procedure: 1. 0.8% of Agarose, Bacteriological, Bacto, and Agar C were prepared.8 2. Agars were heated using a microwave until they completely liquefied. 3. Using the Logger Pro temperature probe, when the temperature dropped to 60℃, both the conductivity (both in TDS9 and in µS10) and the pH were recorded and are shown in Chart 1 below. Agars Conductivity pH (±0.05) in TDS(a) (±1) in µS(b) (±1) Agarose 669 335 8.54Bacteriological Agar 826 412 8.48 Bacto Agar 878 440 8.49 Agar C 905 454 8.40 Chart 1: conductivity and pH of various agars (a) TDS is a unit for total dissolved solids that include both organic and inorganic substances in a liquid (b) µS is a unit for electric conductance due to NaCl7 Software used to collect data using probes electronically8 Refer to Appendix 1 for detailed procedure.9 Unit for total dissolved solids that include both organic and inorganic substances in a liquid10 Unit for electric conductance due to NaCl Page 9 / 32
    • Results of the preliminary investigationAccording to this preliminary investigation, Agarose, which is the optimal medium for gelelectrophoresis, contains the least ions compared to other substitute agars. BacteriologicalAgar contains the next least ions among the substitutes, which supports the stance thatBacteriological Agar can be a viable substitute. pH was all consistent, around 8.5, whichindicates that gel electrophoresis is possible, because TBE buffer works best at pH of 8. Page 10 / 32
    • 4.0 Lambda HindIII DNA Ladder on 0.8% AgaroseProcedure 1) 0.8% Agarose was prepared, heated and poured to make the gel. 2) 10µl of Lamda DNA HindIII Ladder and 5µl of the loading dye were loaded in the well. 3) Gel electrophoresis was performed at 50V for three hours and stained using the Methylene Blue.Data CollectionAfter the gel was de-stained using distilled water, the distances of the fragments from thewell were measured using a ruler. The HindIII ladder is shown in Figure 4.Qualitative DataAccording to Figure 4 below, six fragments were observed. The first three fragments werevery noticeable, but the latter three fragments were not easily detected, because the latterthree consist of smaller amount of DNA. Figure 4: HindIII Ladder on 0.8% Agarose gel. Page 11 / 32
    • Quantitative DataThe distances of the fragments from the well were measured and recorded in Chart 2 below.The literature values for each base pair were used. 0.8% Agarose HindIII Ladder Base Pair/ bp Distance from the well/ cm 23130 13 9416 18 6557 21 4361 25 2322 37 2027 40 Chart 2: HindIII Ladder and the fragments’ distances from the well on 0.8% Agarose HindIII Ladder on 0.8% Agarose 23130 10000 Base Pairs/ bp 9416 6557 4361 2322 2027 1000 10 15 20 25 30 35 40 45 Distance from the well/ cm Graph 1: HindIII Ladder on 0.8% Agarose mediumThe data was plotted using the semi log graph on Graph 1 above, because the rate of DNAfragment migration is inversely proportional to the length of the fragment. From the datapoints, when given an unknown sample, the base pair can be interpolated. Page 12 / 32
    • 5.0 Methodology for Choosing the Agar5.1 Modifying and Facilitating the Gel SlabsSince it is crucial to run 4 different kinds of agar simultaneously to make sure that thecontrolled variables are consistent, modification to the gel slab was vital. The original gelslab only allowed one kind of agar with multiple wells. Thus, by dividing the gel slabs, agarswere not wasted, and comparison was done efficiently.An acrylic plastic is cut with the same height and length as the sides of the original gel slab. Itwas measured using rulers for exactness and cut using the sharp blade. When the pieces werecut identically, they were glued to the original gel slab using the super glue, which mostlyconsists of ethyl cyanoacrylate11.Then, the modified slabs, shown in Figure 5, were tested with distilled water to check theleakage. Even though it prevented most of the leakage, water leaked to the bottom. So, duringthe real experiment, the gels had to be poured very carefully at lower temperatures, at whichthe gels start to harden. Figure 5: modified gel slabs11 Ethyl cyanoacrylate is a transparent liquid with low viscosity that is a main component of super glue. Page 13 / 32
    • 5.2 MethodologiesLambda DNA that was digested with HindIII restriction enzyme was used. It consisted of twomarkers, which were Bromophenol Blue12 in purple and Xylene Cyanol13 in sky blue. Sincethis experiment is designed to select the agar with high resolution, all of the agars wereprepared with the same concentration, 0.8%.5.2.1 Preparation of Gels for Four Different Agars1. 20x TBE buffer is diluted to 1x TBE buffer.2. 0.8% agars were prepared as shown in Figure 6 below.3. The mixture from step 2 in conical flask was capped with a small beaker to prevent water loss and heated in the microwave until it boiled4. The solution was cooled and swirled to keep the solution homogenous.5. Steps 2 to 5 are repeated with Bacto, Bacteriological, and Agar C.6. The agar solutions are poured into the gel slab carefully to prevent possible leakage and the well comb was placed until the solutions solidified.. Figure 6: gel preparation process12 Negatively charged color marker that migrates at the same pace as 500bp DNA13 Negatively charged color marker that migrates at the same pace as 4000bp DNA Page 14 / 32
    • 5.2.2 Preparation of Lambda DNA Samples1. 15µl of HindIII-digested Lambda DNA and 5µl of loading dye, sucrose solution, were mixed with a pulse.2. Step 1 was repeated to make 4 identical samples.5.2.3 Preparation of Loading and Gel Electrophoresis1. The samples were loaded into the middle wells of carefully using micropipette.2. 1x TBE buffer was transferred to the chamber to entirely cover the gel slab.3. The gels were electrophoresed, using 50V, until the Bromophenol Blue marker reached the end of the gel slab as shown in Figure 7 below. Figure 7: gel electrophoresis process Page 15 / 32
    • 5.2.4 Staining and Documenting (Summarized in Figure 8 below)1. Gels are placed in petri dishes and Methylene Blue14 is poured to cover the entire surface.2. After 20 minutes, the gels are transferred to larger container and de-stained until the DNA fragments became visible.3. Gels are transferred to acryl board on top of white background and documented using camera under direct light. Figure 8: staining and de-staining process and documenting process14 Refer to appendix 2 for its selection as the main staining dye Page 16 / 32
    • 5.3 Data CollectionThree different agars were tested with Agarose as the control. As shown in Figure 9 below,only Agarose was de-stained completely while the others were heavily stained. Figure 9: contrasted results of Agarose, Bacto, Bacteriological, and Agar C.5.4 Comparison on Bacto and Bacteriological Agar with Different Concentration.To find out optimal type, Bacto and Bacteriological Agar were tested with differentconcentration because both resolved three fragments with 0.8% concentration. Agar C waseliminated, because it resolved only two fragments. The variation in concentration, 0.6% and0.8%, could help successfully choosing the better agar, as shown in Figure 10 below. Bacto Bacto Bacteriological Bacteriological 0.6% 0.8% 0.6% 0.8% Figure 10: contrasted results of 0.6% and 0.8% Bacto and Bacteriological Agar. Page 17 / 32
    • 5.5 Data AnalysisGenerally, fragments in 0.8% Agarose migrated faster than those in other mediums. Thissuggests that even if alternative agar with best resolution was found, it is necessary to modifyconcentrations to produce comparable results to those of Agarose. From the above result,Bacto, Bacteriological, and Agar C could resolve three, three, and two clear fragmentsrespectively. Even Agarose could not show the last three fragments clearly, so it seems likelythat Bacto and Bacteriological Agar can produces similar results as those of Agarose.Qualitatively, Agarose became almost colorless after de-staining process, while other agarswere still stained and dark. Especially, Bacto and Agar C were heavily colored. In could beinferred that since only the bottom parts are purple, the agars either contained too muchnegatively charged ions that were stained by the Methylene Blue or allowed BromophenolBlue to diffuse, which could not be de-stained quickly. In either case, the coloring might haveimpeded the fragments to be visible.Figure 10 shows that while both 0.6% and 0.8% Bacteriological Agar could resolve threeclear fragments, 0.6% and 0.8% Bacto Agar could resolve one and three clear fragmentsrespectively. Based on observation, the data also supports that Bacteriological Agar is clearerand homogenous in terms of color, which can aid the visibility of fragments.Thus, Bacteriological Agar was chosen for further experiment, because it showed relativelyhigh resolution and produced clear background after the de-staining process. Since the DNAfragments migrated at different pace depending on the gel’s concentration, changing theconcentration to produce more comparable results was essential. Page 18 / 32
    • 6.0 Methodology for Testing Different Concentrations of Bacteriological Agar, which was chosen from the previous experiments6.1 MethodologiesSince this part of experiment is designed to find out the optimum concentration, 0.6%, 0.8%,and 1.0% Bacteriological Agar were tested. Also, to eliminate possible limitations and errorsresulting from the use of two markers, the Lambda DNA was directly treated with HindIIIrestriction enzyme in the incubator. Since the starting marker is essential to know the pace ofDNA fragments migration, a small amount of Bromophenol Blue was only applied toAgarose, which could still produce clear results in the presence of markers.6.1.1 Preparation of Gels with Different Concentrations1. 0.8% Agarose solution was prepared.2. 0.6%, 0.8%, and 1.0% Bacteriological Agar solutions were prepared.3. Solutions made from steps 1 and 2 were heated in the microwave until they boiled.4. The solutions were cooled and swirled to keep the solutions homogenous.5. The agar solutions were poured into the gel slab carefully to prevent possible leakage and the well comb was placed until the solutions solidified.6.1.2 Preparation of Lambda DNA Sample Digested with HindIII1. 32µl of Lambda DNA, 20µl of Buffer 2 and 4µl of HindIII restriction enzyme were mixed using the pulse in a micro centrifuge.2. The solution was incubated at 37˚C for more than 24 hours to ensure complete reaction.3. 14µl was transferred for each 4 micro centrifuge tube and 5µl of loading dye was added to each tube.4. 3µl of diluted Bromophenol Blue was loaded to 0.8% Agarose gel only. Page 19 / 32
    • 6.2 Data CollectionThree trials were performed to obtain precise data as shown in Figure 11, 12, and 13 andstained with Methylene Blue. After documenting, the trials were contrasted to make thefragments clearer. Figure 11: the first trial Figure 12: the second trial Figure 13: the third trial Page 20 / 32
    • 6.3 Data AnalysisOnly three fragments were clearly visible in standard 0.8% Agarose. This may be due toenzyme malfunctioning or staining errors. Even if the DNA with HindIII were left in anincubator more than enough time for complete enzyme reaction, if the enzyme ismalfunctioning, then it may not have digested the DNA properly. Another possibility is thatMethylene Blue staining was an inadequate method. When the DNA fragments are very short,such as 512bp and 125bp, they are not obviously visible, because Methylene Blue cannotsuccessfully stain small fragments to be visible. Therefore, it can only be assumed that therest of the DNA fragments follow the same pace as the three visible fragments.The three trials show that both 0.6% and 0.8% Bacteriological Agar are capable of separatingthe three fragments clearly, whereas 1.0% Bacteriological Agar could only produce one ortwo clear fragments. This is because the pores were too close together or too small thatobstruct the fragments from migrating. When comparing the DNA fragments’ locations, 0.6%Bacteriological Agar was more comparable than 0.8% Bacteriological Agar to 0.8% Agarose.This suggests that the pores of 0.6% Bacteriological Agar and of 0.8% Agarose are similar insize and structure.It seems likely that the concentration determines the pore sizes and structures. When theconcentration increases, the pore sizes decreases and the structure becomes more organized,which hinders the DNA fragments from migrating efficiently. Perhaps, the higherconcentration of ions and impurities in Bacteriological Agar add onto the pores as obstacles. Page 21 / 32
    • Figure 14: diagram of pores and ions and impurities in 0.8% Agarose, 0.8% BacteriologicalAgar, and 0.6% Bacteriological AgarFrom the Figure 14 above, it can be justified that the unnecessary ions and impurities impedethe pathway for the DNA fragments movement. Thus, it confirms that 0.6%Bacteriological Agar that has larger pores and less concentration of ions and impuritiesthan 0.8% Bacteriological Agar can perform just like 0.8% Agarose. Page 22 / 32
    • 7.0 Further Investigation on Resolving More Fragments, using a Different StainThe same procedure is used, but the gels were stained using the QUIKView DNA Stain15.After documenting, images were contrasted to show the fragments more clearly. Themeasurements were taken by measuring the distance from the well.Qualitative DataSix fragments were visible in 0.8% Agarose, while only four and three fragments were visiblein 0.6% and 0.8% Bacteriological Agar respectively. As show in Figure 15, bothbacteriological agars were heavily stained on the bottom due to the migration of charged ions.0.6% Bacteriological Agar could resolve four fragments and the locations of the fragmentswere comparable to those of Agarose. However, the fragments in 0.8% Bacteriological Agarcould not be resolved well. Figure 15: HindIII Ladder on 0.8% Agarose, 0.6% and 0.8% Bacteriological Agar15 QUIKView DNA Stain is the new DNA stain to be tested that is manufactured from Carolina company Page 23 / 32
    • Quantitative DataThe distances of the fragments from the well were measured using a ruler and recorded on theChart 3 below. The distances of 0.6% Bacteriological Agar was more comparable than 0.8%Bacteriological Agar to 0.8% Agarose. 0.6% 0.8% 0.8% Agarose Bacteriological Agar Bacteriological Agar HindIII Ladder Distance from the well/ cm Base Pair(a)/ bp 23130 34 36 30 9416 39 42 34 6557 43 45 36 4361 45 52 -(b) 2322 57 - - 2027 63 - -Chart 3: HindIII Ladder and the fragments’ distances from the well on 0.8% Agarose, 0.6%and 0.8% Bacteriological Agar (a) The literature values of the HindIII Ladder on Figure 1 were used. (b) – represents that the fragment was not visible for recording. Page 24 / 32
    • Data points from Chart 3 were plotted using a semi log graph to obtain linear trends, shown in Graph 2 below. HindIII Digest on 0.8% Agarose 0.6% Bacteriological Agar 0.8% Bacteriological Agar Different Agar Mediums 23130 Base Pair/ bp 9416 6557 4361 2322 2027 25 30 35 40 45 50 55 60 65 Distance from the well/ cm Graph 2: Comparison of Hind III Ladder on standard 0.8% Agarose and 0.6% and 0.8% Bacteriological Agar. Page 25 / 32
    • 8.0 Discussion of the ResultsThe experiments using the QUIKView DNA stain showed that 0.6% Bacteriological Agar ismore comparable than 0.8% Bacteriological Agar to 0.8% Agarose. Based on Graph 2, thegap between 0.6% Bacteriological Agar and 0.8% Agarose is smaller than that between 0.8%Agarose and 0.8% Bacteriological Agar. For accuracy, percentage errors were calculated andshown in Chart 4. 0.6% Bacteriological Agar 0.8% Bacteriological AgarHindIII Ladder Base Pair/ bp Percentage Errors/ % 23130 9416 6557 4361 - (a) Chart 4: percentage error calculations (a) The percent error cannot be calculated because the data is missing.0.6% Bacteriological Agar consists of lower percent errors than 0.8%. This confirms that 0.6%is a better substitute than 0.8% Bacteriological Agar.0.6% Bacteriological Agar could resolve fragments ranging from 23130bp to 4361bp. Thus, itis only comparable to 0.8% Agarose when the fragments are in that range. Given an unknownsample, the base pair of the sample can be interpolated using the 0.6% Bacteriological Agargraph. However, it is not suitable when the fragments are smaller than 4361bp.In short, both experiments using the Methylene Blue and QUIKView DNA stain, 0.6%Bateriological Agar produced better results than 0.8% Page 26 / 33
    • 9.0 EvaluationDuring gel preparation, since the handmade gel slabs were fragile compared to the original.Thus, the gels had to be poured very carefully to prevent leakage. Even though the solutionwas swirled to ensure that the gel is homogeneous, it is possible that the poured gel is morediluted than the originally prepared gel, because of the slow pouring process.The staining process also had some difficulties, because agars de-stained at different paces.Agarose was easily de-stained while Bacteriological Agar took more time. Due to thisinconsistency, the gels had to be de-stained separately.9.1 Limitations and UncertaintiesOnly two kinds of stains were available, Methylene Blue and QUIKView DNA stain. WhileMethylene Blue stained three fragments, QUIKView DNA stain stained four fragmentssuccessfully. Hence, the results depended on the staining method. Other stains could not betested due to time constraint.Also, de-staining method needed to be modified. For agars with high ion and impurityconcentrations, the bottom part of the agar blocks could not be successfully de-stained, whichhindered viewing the fragments. When de-stained for longer time, the DNA was de-stained aswell. Since it is impossible to de-stain the upper and the lower part separately using thecurrent method, better method is needed.The Bromophenol Blue was used as the loading dye, which diffused into the agar gel whenthe gel is left for long time. This could have hindered viewing the fragments as well, since thecolor resembled the stain.The solution was heated in the microwave, which the temperature cannot be measured andwas not constant. To ensure that the solution become completely liquefied and homogenous, Page 27 / 32
    • evaporation was inevitable, which can alter the concentration.9.2 Ways of ImprovementsDifferent stains can be purchased and tested to find the optimal stain. Since the stainingmethod can alter the outcome, this improvement is crucial. For example the fluorescenttagging method, which require Ethidium Bromide (EtBr) and ultraviolet light, may capture allfragments. For this experiment, EtBr could not be used, because it is known as a carcinogen.For a better de-staining method, ion exchange resin is needed. Since the stain colors all thenegatively charged particles, gels with more anions and impurities are heavily colored,making the de-staining process difficult. Hence, anion exchange is needed to remove excessanions.Instead of using Bromophenol Blue, using Cresol Red or Orange G can improve the overallobservation of the gel, because those loading dyes are very distinct from the color of the stain.Since different types of agars have different melting points, preparing a water bath adjusted tothe agar’s melting point can slowly liquefy the solution without abrupt evaporations. Eventhough this will take a longer time, it ensures that there is no water loss. Page 28 / 32
    • 9.3 Unresolved Questions for Further ResearchThroughout the experiment, only Lambda DNA, which is highly purified and expensive, isused. However, it is necessary to figure out if Bacteriological Agar can resolve fragments ofother, preferably cheaper, DNAs, because the rationale of this investigation is to make the gelelectrophoresis process cost-effective. Hence, testing onion DNA or bacteria DNA is essentialto make the process manageable at underprivileged institutes.Moreover, different types of restriction enzymes can be tested to ensure that BacteriologicalAgar is a viable means for other enzymes and base pairs. For example, BamHI16 is extremelydifficult to observe with low quality gel, because the fragments are so close together. Sincethe experiment only dealt with HindIII, testing other enzymes will improve this investigation.Through testing them, the base pair range of Bacteriological Agar resolution, shorter than4361bp, can also be found out.Throughout the experiment, all electrophoresis was done under 50V. Voltage is directlyrelated to the separation of DNA fragments and migration pace, so different voltages havedifferent effects on the results. Thus, with a more advanced chamber, optimum voltage can befound.16 A restriction enzyme that digests the DNA at four different sites and produces five fragments of similarlengths Page 29 / 32
    • 10.0 ConclusionThe series of experiments shows that 0.6% Bacteriological Agar can be a suitable substitutefor 0.8% Agarose. The first part of the experiment, which dealt with the resolution ofdifferent agars, shows that Bacteriological Agar has relatively high resolution compared toother types of agars, yet has lower quality than Agarose. When different concentrations weretested, 0.6% Bacteriological Agar could produce similar DNA fragments positions as those in0.8% Agarose.It is also verified that the lower concentration produce larger pores, which allow for fasterDNA fragments migration pace, under constant voltage. Also, lower concentration containslesser impurities, which aids the clarity after de-staining process. The hypothesis was correct;high ion concentration impeded the clarity as the stain colored the negatively chargedparticles, making the gels denser in color, especially the bottom parts.So far, the best substitute is 0.6% Bacteriological Agar. However, further research isneeded to verity that it is a suitable substitute. In terms of this set of experiment, 0.6%Bacteriological Agar is a cost-effective, yet viable means for gel electrophoresis. Page 30 / 32
    • 11.0 AppendixAppendix 1Preparation of 0.8% agar solution 1) 0.8g of agar powder was accurately weighed using the electronic weighing balance 2) 0.8g of agar and 100cm3 of TBE buffer were transferred to a conical flaskAppendix 2Choosing the staining dye.In dying the gel, a positively charged color stain or a fluorescent tag using ultraviolet lightcan be used. However, since the fluorescent tag method requires Ethidium Bromide, which isknown as carcinogen, only color staining methods are considered. Two options for color stainwere Methylene Blue and Carolina BluTM. Below shows a sample run using pre-digestedHindIII ladder. (a) (b) (c) (d) (a) Methylene Blue with 10µl DNA (b) Methylene Blue with 10µl DNA (c) Carolina BluTM with 10µl DNA (d) Methylene Blue with 5µl DNABased on this sample, Methylene Blue de-stained faster and the contrast between the DNAfragments and the gel was more conspicuous. Also, Methylene Blue could stain lesser amountof DNA well. Thus, Methylene Blue was chosen over Carolina BluTM. Page 31 / 32
    • 12.0 Bibliography[1] "Restriction of Lambda DNA." Science Education Program. Web. 14 July 2010. <http://education.llnl.gov/bep/science/10/sLamb.html>.[2] "Phage Lambda DNA Hind III Digest Ready-to-use - GeneON: Products for Molecular Biology." GeneON - GeneON: Products for Molecular Biology. Web. 14 July 2010. <http://www.taq-dna.com/phage-lambda-dna-hind-iii-digest-ready-to-use- _137.html>.[3] "Phage Lambda DNA Hind III Digest Ready-to-use - GeneON: Products for Molecular Biology." GeneON - GeneON: Products for Molecular Biology. Web. 14 July 2010. <http://www.taq-dna.com/phage-lambda-dna-hind-iii-digest-ready-to-use- _137.html>.[4] 华桥-Fargarose琼脂糖. Web. 14 Sept. 2010. <http://www.sunmabio.com/eng/fargarose.htm>.[5] "Lambda DNA - HindIII Digest." Southern Biological. Web. <http://www.southernbiological.com/Assets/pdf/Products/Kits&Equipment/GelElectr ophoresis/G42_60InfoSheet.pdf>. Page 32 / 32