IB EE on DNA gel electrophoresis using different agar medium by lawrence kok

Research Question: Can Bacteriological Agar be a suitable substitute for Agarose in terms 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

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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.

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Abstract DNA gel electrophoresis is a process of separating DNA fragments using an electric field and a gel matrix. Negatively charged DNA fragments move to the positive end and the gel matrix is used as a medium for migration. Because of the gel’s pores, the shortest DNA fragment moves faster than the longest DNA fragment, separating the fragments according to their sizes. Agarose is commonly used, because it is extremely refined, containing the minimum amount 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 for comparable resolution. Lambda DNA was used to perform gel electrophoresis. When choosing the best agar among three alternatives, restriction enzyme digested DNA HindIII and ionized markers were used. Bacteriological Agar had the best resolution among the alternatives. Hence modifications on the concentration were performed with Bacteriological Agar to find the optimal concentration for it to produce comparable results to those by Agarose. In this part of the experiment, DNA was digested using the enzyme directly and the mixture was incubated for more than 24 hours for complete enzyme reaction 0.6%, 0.8%, and 1.0% Bacteriological Agars were tested and compared to 0.8% Agarose. In both processes, the gel matrices were stained using Methylene Blue. For further experiment, QUIKView DNA stain was used. Results showed that Bacteriological Agar produced the best resolution. Of different concentrations, 0.6% Bacteriological Agar produced the most comparable results to those of Agarose. 0.6% Bacteriological Agar could separate multiple fragments well and their locations were similar to those in Agarose. Methylene Blue stained three fragments on both 0.6% 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)

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Table of Contents Acknowledgement .................................................................................................................... 1 Abstract ..................................................................................................................................... 2 1.0 Introduction ........................................................................................................................ 5 1.1 Background Information ...................................................................................................... 5 1.2 Rationale of Study................................................................................................................ 6 2.0 Hypothesis........................................................................................................................... 8 3.0 Preliminary Investigation on Different Types of Agar ................................................. 9 4.0 Lambda HindIII DNA Ladder on 0.8% Agarose ......................................................... 11 5.0 Methodology for Choosing the Agar .............................................................................. 13 5.1 Modifying and Facilitating the Gel Slabs .......................................................................... 13 5.2 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 ............................................................................ 16 5.3 Data Collection .................................................................................................................. 17 5.4 Comparison on Bacto and Bacteriological Agar with Different Concentrations .............. 17 5.5 Data Analysis ..................................................................................................................... 18 6.0 Methodology for Testing Different Concentrations of Bacteriological Agar, which was chosen from the previous experiments ......................................................................... 19 6.1 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..................................... 19 6.2 Data Collection .................................................................................................................. 20 6.3 Data Analysis ..................................................................................................................... 21 7.0 Further Investigation on Resolving Six Fragments, using a Different Stain ..................... 23 8.0 Discussion of the Results ................................................................................................. 26 9.0 Evaluation ......................................................................................................................... 27 9.1 Limitations and Uncertainties ............................................................................................ 27 9.2 Ways of Improvements ...................................................................................................... 28 9.3 Unresolved Questions and Further Research ..................................................................... 29 10.0 Conclusion ....................................................................................................................... 30 11.0 Appendix .......................................................................................................................... 31 12.0 Bibliography .................................................................................................................... 32

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1.0

Introduction

1.1

Background Information

Gel electrophoresis is a biotechnology used to separate DNA fragments according to its length using agar medium and electric field. Since DNA is negatively charged because of phosphate group, it moves to the positive pole of the electric field. As the fragments move through the pores of agar medium, the shorter fragments move faster than the longer fragments. 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 7 fragments 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 2

Double-stranded linear DNA from virus particle called Lambda phage 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 the medium 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 electric field when submerged in TBE buffer4. Also, it de-stains quickly after stained with Methylene Blue5. Since Methylene Blue stains all of the negatively charged ions including DNA, gels with high ion concentration will appear denser in color and will take longer time to de-stain.

1.2

Rationale of Study

Gel electrophoresis is commonly taught in high school as well as in colleges. However, often times, not all schools promote the experiment, because of its cost, because Agarose is very expensive compared to other less-purified agars. The gel electrophoresis chamber itself costs between $150 to $300 and Agarose ranges from $125.28 to $360.04 while Bacteriological Agar costs only around $20.08. Not only that, other materials and equipments such as Lambda DNA, restriction enzymes, micropipettes, and others cost quite a lot as well.

This practical skill is somewhat a costly experiment for high school students, because

Linear polysaccharide polymer of agarobiose TBE stands for Tris Borate EDTA and it deprotonates DNA, contains necessary ions, and protects nucleic acids. 5 A positively charged dye used for staining DNA fragments in the gel 4

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Agarose, DNA, restriction enzymes, and other reagents are not recyclable. In order to devise a cheaper method, cost-effective agar substitute must be found. Thus, finding alternative, yet viable substitute will be vital to reduce the economical burden of institutions and to give more students chance to perform such experiment.

Moreover, DNA electrophoresis is often used to determine the size of unknown DNA fragment by comparing it with DNA ladder6. This is also used at crime scenes. HindIII restriction enzyme ideally produces eight fragments of various lengths. By comparing HindIII ladder with unknown DNA, a possible length of unknown DNA can be interpolated. Since they are run with Agarose, a viable substitute will enable such DNA profiling processed economically.

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 DNA fragments clearly, comparing the resolution was essential. Moreover, the pace of the DNA fragment migration is inversely proportional to the concentration of the gel. By changing the concentrations, the substitute for Agarose can be found, which is much cheaper, yet can produce comparable results.

Thus, the research question is Can Bacteriological Agar be a suitable substitute for Agarose 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 with those obtained from Agarose medium?

DNA ladder consists of many DNA fragments of different lengths Page 7 / 32

2.0

Hypothesis

Since 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 the gel contains the least amount of ions, like the controlled Agarose, so that it can facilitate the process and produce results with high resolution.

When qualitatively observed, the particles of Agarose were very fine and white as seen in Figure 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 Agar appeared yellower and composed of relatively larger particles, which suggest that it may form irregular pores. Agar C had fine particles, but was heavily colored. Hence, Bacteriological Agar may be a better substitute.

Figure 3: different agar particles When comparing the liquefied Agarose and Bacteriological Agar, Bacteriological Agar seemed more viscose than Agarose. In higher gel concentration, the fragments migrate at a relatively slow pace, so the separation of smaller fragments are conspicuous, whereas in lower gel concentration the fragments move at fast pace and the separation of larger fragments are more visible. The rate of migration is inversely proportional to the concentration. Moreover, the resolution, or the intensity of fragments, depends on the amount of 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 is hypothesized that Bacteriological Agar can be a suitable substitute for Agarose in terms of DNA gel electrophoresis use.

3.0

Preliminary Investigation on Different Types of Agar

A preliminary investigation was done to research the correlations between the conductivity and pH and the resolution. All of the agars were tested using the Logger Pro7 conductivity probe 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.

Conductivity

Agars

pH (±0.05)

in TDS(a) (±1)

in µS(b) (±1)

Agarose

669

335

8.54

Bacteriological 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 NaCl 7

Software used to collect data using probes electronically Refer to Appendix 1 for detailed procedure. 9 Unit for total dissolved solids that include both organic and inorganic substances in a liquid 10 Unit for electric conductance due to NaCl 8

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Results of the preliminary investigation According to this preliminary investigation, Agarose, which is the optimal medium for gel electrophoresis, contains the least ions compared to other substitute agars. Bacteriological Agar contains the next least ions among the substitutes, which supports the stance that Bacteriological Agar can be a viable substitute. pH was all consistent, around 8.5, which indicates that gel electrophoresis is possible, because TBE buffer works best at pH of 8.

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4.0

Lambda HindIII DNA Ladder on 0.8% Agarose

Procedure 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 Collection After the gel was de-stained using distilled water, the distances of the fragments from the well were measured using a ruler. The HindIII ladder is shown in Figure 4.

Qualitative Data According to Figure 4 below, six fragments were observed. The first three fragments were very noticeable, but the latter three fragments were not easily detected, because the latter three consist of smaller amount of DNA.

Figure 4: HindIII Ladder on 0.8% Agarose gel.

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Quantitative Data The 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

9416

6557

4361

2322

2027

Chart 2: HindIII Ladder and the fragments’ distances from the well on 0.8% Agarose

HindIII Ladder on 0.8% Agarose

Base Pairs/ bp

23130

10000

9416 6557 4361 2322

2027

1000 10

Distance from the well/ cm

Graph 1: HindIII Ladder on 0.8% Agarose medium The data was plotted using the semi log graph on Graph 1 above, because the rate of DNA fragment migration is inversely proportional to the length of the fragment. From the data points, when given an unknown sample, the base pair can be interpolated.

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5.0

Methodology for Choosing the Agar

5.1

Modifying and Facilitating the Gel Slabs

Since it is crucial to run 4 different kinds of agar simultaneously to make sure that the controlled variables are consistent, modification to the gel slab was vital. The original gel slab only allowed one kind of agar with multiple wells. Thus, by dividing the gel slabs, agars were 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. It was measured using rulers for exactness and cut using the sharp blade. When the pieces were cut identically, they were glued to the original gel slab using the super glue, which mostly consists of ethyl cyanoacrylate11.

Then, the modified slabs, shown in Figure 5, were tested with distilled water to check the leakage. Even though it prevented most of the leakage, water leaked to the bottom. So, during the real experiment, the gels had to be poured very carefully at lower temperatures, at which the gels start to harden.

Figure 5: modified gel slabs

Ethyl cyanoacrylate is a transparent liquid with low viscosity that is a main component of super glue. Page 13 / 32

5.2

Methodologies

Lambda DNA that was digested with HindIII restriction enzyme was used. It consisted of two markers, which were Bromophenol Blue12 in purple and Xylene Cyanol13 in sky blue. Since this experiment is designed to select the agar with high resolution, all of the agars were prepared with the same concentration, 0.8%.

5.2.1

Preparation of Gels for Four Different Agars

20x TBE buffer is diluted to 1x TBE buffer.

0.8% agars were prepared as shown in Figure 6 below.

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 boiled

The solution was cooled and swirled to keep the solution homogenous.

Steps 2 to 5 are repeated with Bacto, Bacteriological, and Agar C.

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 process

12 13

Negatively charged color marker that migrates at the same pace as 500bp DNA Negatively charged color marker that migrates at the same pace as 4000bp DNA Page 14 / 32

5.2.2

Preparation of Lambda DNA Samples

15µl of HindIII-digested Lambda DNA and 5µl of loading dye, sucrose solution, were mixed with a pulse.

Step 1 was repeated to make 4 identical samples.

5.2.3 Preparation of Loading and Gel Electrophoresis 1.

The samples were loaded into the middle wells of carefully using micropipette.

1x TBE buffer was transferred to the chamber to entirely cover the gel slab.

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

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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.

After 20 minutes, the gels are transferred to larger container and de-stained until the DNA fragments became visible.

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 process

Refer to appendix 2 for its selection as the main staining dye Page 16 / 32

5.3

Data Collection

Three 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 different concentration because both resolved three fragments with 0.8% concentration. Agar C was eliminated, because it resolved only two fragments. The variation in concentration, 0.6% and 0.8%, could help successfully choosing the better agar, as shown in Figure 10 below.

Bacto 0.6%

Bacto 0.8%

Bacteriological 0.6%

Bacteriological 0.8%

Figure 10: contrasted results of 0.6% and 0.8% Bacto and Bacteriological Agar.

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5.5

Data Analysis

Generally, fragments in 0.8% Agarose migrated faster than those in other mediums. This suggests that even if alternative agar with best resolution was found, it is necessary to modify concentrations to produce comparable results to those of Agarose. From the above result, Bacto, Bacteriological, and Agar C could resolve three, three, and two clear fragments respectively. Even Agarose could not show the last three fragments clearly, so it seems likely that Bacto and Bacteriological Agar can produces similar results as those of Agarose.

Qualitatively, Agarose became almost colorless after de-staining process, while other agars were still stained and dark. Especially, Bacto and Agar C were heavily colored. In could be inferred that since only the bottom parts are purple, the agars either contained too much negatively charged ions that were stained by the Methylene Blue or allowed Bromophenol Blue to diffuse, which could not be de-stained quickly. In either case, the coloring might have impeded the fragments to be visible.

Figure 10 shows that while both 0.6% and 0.8% Bacteriological Agar could resolve three clear fragments, 0.6% and 0.8% Bacto Agar could resolve one and three clear fragments respectively. Based on observation, the data also supports that Bacteriological Agar is clearer and homogenous in terms of color, which can aid the visibility of fragments.

Thus, Bacteriological Agar was chosen for further experiment, because it showed relatively high resolution and produced clear background after the de-staining process. Since the DNA fragments migrated at different pace depending on the gel’s concentration, changing the concentration to produce more comparable results was essential.

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6.0

Methodology for Testing Different Concentrations of Bacteriological Agar, which was chosen from the previous experiments

6.1

Methodologies

Since 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 errors resulting from the use of two markers, the Lambda DNA was directly treated with HindIII restriction enzyme in the incubator. Since the starting marker is essential to know the pace of DNA fragments migration, a small amount of Bromophenol Blue was only applied to Agarose, which could still produce clear results in the presence of markers.

6.1.1

Preparation of Gels with Different Concentrations

0.8% Agarose solution was prepared.

0.6%, 0.8%, and 1.0% Bacteriological Agar solutions were prepared.

Solutions made from steps 1 and 2 were heated in the microwave until they boiled.

The solutions were cooled and swirled to keep the solutions homogenous.

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 HindIII 1.

32µl of Lambda DNA, 20µl of Buffer 2 and 4µl of HindIII restriction enzyme were mixed using the pulse in a micro centrifuge.

The solution was incubated at 37˚C for more than 24 hours to ensure complete reaction.

14µl was transferred for each 4 micro centrifuge tube and 5µl of loading dye was added to each tube.

3µl of diluted Bromophenol Blue was loaded to 0.8% Agarose gel only.

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6.2 Data Collection Three trials were performed to obtain precise data as shown in Figure 11, 12, and 13 and stained with Methylene Blue. After documenting, the trials were contrasted to make the fragments clearer.

Figure 11: the first trial

Figure 12: the second trial

Figure 13: the third trial Page 20 / 32

6.3 Data Analysis Only three fragments were clearly visible in standard 0.8% Agarose. This may be due to enzyme malfunctioning or staining errors. Even if the DNA with HindIII were left in an incubator more than enough time for complete enzyme reaction, if the enzyme is malfunctioning, then it may not have digested the DNA properly. Another possibility is that Methylene 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 cannot successfully stain small fragments to be visible. Therefore, it can only be assumed that the rest 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 separating the three fragments clearly, whereas 1.0% Bacteriological Agar could only produce one or two clear fragments. This is because the pores were too close together or too small that obstruct 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 in size and structure.

It seems likely that the concentration determines the pore sizes and structures. When the concentration increases, the pore sizes decreases and the structure becomes more organized, which hinders the DNA fragments from migrating efficiently. Perhaps, the higher concentration of ions and impurities in Bacteriological Agar add onto the pores as obstacles.

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Figure 14: diagram of pores and ions and impurities in 0.8% Agarose, 0.8% Bacteriological Agar, and 0.6% Bacteriological Agar From the Figure 14 above, it can be justified that the unnecessary ions and impurities impede the pathway for the DNA fragments movement. Thus, it confirms that 0.6% Bacteriological Agar that has larger pores and less concentration of ions and impurities than 0.8% Bacteriological Agar can perform just like 0.8% Agarose.

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7.0

Further Investigation on Resolving More Fragments, using a Different Stain

The 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. The measurements were taken by measuring the distance from the well.

Qualitative Data Six fragments were visible in 0.8% Agarose, while only four and three fragments were visible in 0.6% and 0.8% Bacteriological Agar respectively. As show in Figure 15, both bacteriological 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 fragments were comparable to those of Agarose. However, the fragments in 0.8% Bacteriological Agar could not be resolved well.

Figure 15: HindIII Ladder on 0.8% Agarose, 0.6% and 0.8% Bacteriological Agar

QUIKView DNA Stain is the new DNA stain to be tested that is manufactured from Carolina company Page 23 / 32

Quantitative Data The distances of the fragments from the well were measured using a ruler and recorded on the Chart 3 below. The distances of 0.6% Bacteriological Agar was more comparable than 0.8% Bacteriological Agar to 0.8% Agarose.

0.8% Agarose HindIII Ladder Base Pair(a)/ bp

0.6% 0.8% Bacteriological Agar Bacteriological Agar Distance from the well/ cm

23130

9416

6557

4361

-(b)

2322

2027

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.

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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

Base Pair/ bp

23130

9416

6557

4361

2322 2027 25

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.

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8.0

Discussion of the Results

The experiments using the QUIKView DNA stain showed that 0.6% Bacteriological Agar is more comparable than 0.8% Bacteriological Agar to 0.8% Agarose. Based on Graph 2, the gap 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 and shown in Chart 4.

HindIII Ladder Base Pair/ bp

0.6% Bacteriological Agar 0.8% Bacteriological Agar 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, it is only comparable to 0.8% Agarose when the fragments are in that range. Given an unknown sample, the base pair of the sample can be interpolated using the 0.6% Bacteriological Agar graph. 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%

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9.0

Evaluation

During 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 solution was swirled to ensure that the gel is homogeneous, it is possible that the poured gel is more diluted 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 this inconsistency, the gels had to be de-stained separately.

9.1

Limitations and Uncertainties

Only two kinds of stains were available, Methylene Blue and QUIKView DNA stain. While Methylene Blue stained three fragments, QUIKView DNA stain stained four fragments successfully. Hence, the results depended on the staining method. Other stains could not be tested due to time constraint.

Also, de-staining method needed to be modified. For agars with high ion and impurity concentrations, the bottom part of the agar blocks could not be successfully de-stained, which hindered viewing the fragments. When de-stained for longer time, the DNA was de-stained as well. Since it is impossible to de-stain the upper and the lower part separately using the current method, better method is needed.

The Bromophenol Blue was used as the loading dye, which diffused into the agar gel when the gel is left for long time. This could have hindered viewing the fragments as well, since the color resembled the stain.

The solution was heated in the microwave, which the temperature cannot be measured and was 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 Improvements

Different stains can be purchased and tested to find the optimal stain. Since the staining method can alter the outcome, this improvement is crucial. For example the fluorescent tagging method, which require Ethidium Bromide (EtBr) and ultraviolet light, may capture all fragments. 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 the negatively charged particles, gels with more anions and impurities are heavily colored, making the de-staining process difficult. Hence, anion exchange is needed to remove excess anions.

Instead of using Bromophenol Blue, using Cresol Red or Orange G can improve the overall observation 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 to the agar’s melting point can slowly liquefy the solution without abrupt evaporations. Even though this will take a longer time, it ensures that there is no water loss.

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9.3

Unresolved Questions for Further Research

Throughout the experiment, only Lambda DNA, which is highly purified and expensive, is used. However, it is necessary to figure out if Bacteriological Agar can resolve fragments of other, preferably cheaper, DNAs, because the rationale of this investigation is to make the gel electrophoresis process cost-effective. Hence, testing onion DNA or bacteria DNA is essential to make the process manageable at underprivileged institutes.

Moreover, different types of restriction enzymes can be tested to ensure that Bacteriological Agar is a viable means for other enzymes and base pairs. For example, BamHI16 is extremely difficult to observe with low quality gel, because the fragments are so close together. Since the experiment only dealt with HindIII, testing other enzymes will improve this investigation. Through testing them, the base pair range of Bacteriological Agar resolution, shorter than 4361bp, can also be found out.

Throughout the experiment, all electrophoresis was done under 50V. Voltage is directly related to the separation of DNA fragments and migration pace, so different voltages have different effects on the results. Thus, with a more advanced chamber, optimum voltage can be found.

A restriction enzyme that digests the DNA at four different sites and produces five fragments of similar lengths Page 29 / 32

10.0

Conclusion

The series of experiments shows that 0.6% Bacteriological Agar can be a suitable substitute for 0.8% Agarose. The first part of the experiment, which dealt with the resolution of different agars, shows that Bacteriological Agar has relatively high resolution compared to other types of agars, yet has lower quality than Agarose. When different concentrations were tested, 0.6% Bacteriological Agar could produce similar DNA fragments positions as those in 0.8% Agarose.

It is also verified that the lower concentration produce larger pores, which allow for faster DNA fragments migration pace, under constant voltage. Also, lower concentration contains lesser 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 charged particles, making the gels denser in color, especially the bottom parts.

So far, the best substitute is 0.6% Bacteriological Agar. However, further research is needed 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.

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11.0

Appendix

Appendix 1 Preparation 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 flask Appendix 2 Choosing the staining dye. In dying the gel, a positively charged color stain or a fluorescent tag using ultraviolet light can be used. However, since the fluorescent tag method requires Ethidium Bromide, which is known as carcinogen, only color staining methods are considered. Two options for color stain were Methylene Blue and Carolina BluTM. Below shows a sample run using pre-digested HindIII 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 DNA Based on this sample, Methylene Blue de-stained faster and the contrast between the DNA fragments and the gel was more conspicuous. Also, Methylene Blue could stain lesser amount of DNA well. Thus, Methylene Blue was chosen over Carolina BluTM.

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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]

[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>.

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