Page 0 / 32

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

Page 1 / 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 2 / 32

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)

Page 3 / 32

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 4 / 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

Page 5 / 32

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 Double-stranded linear DNA from virus particle called Lambda phage 2 Unit for base pair.

Page 6 / 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

3 Linear polysaccharide polymer of agarobiose

4 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

Page 7 / 32

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?

6 DNA ladder consists of many DNA fragments of different lengths

Page 8 / 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 9 / 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 µS

10) 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 8.54

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

8 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

Page 10 / 32

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.

Page 11 / 32

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.

Page 12 / 32

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 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

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.

23130

9416

6557

4361

23222027

1000

10000

10 15 20 25 30 35 40 45

Base

Pairs/

bp

Distance from the well/ cm

HindIII Ladder on 0.8% Agarose

Page 13 / 32

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

11

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

Page 14 / 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

1. 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 boiled

4. 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 process

12

Negatively charged color marker that migrates at the same pace as 500bp DNA 13

Negatively charged color marker that migrates at the same pace as 4000bp DNA

Page 15 / 32

5.2.2 Preparation of Lambda DNA Samples

1. 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 Electrophoresis

1. 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 16 / 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 process

14

Refer to appendix 2 for its selection as the main staining dye

Page 17 / 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.

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

Bacto Bacto Bacteriological Bacteriological

0.6% 0.8% 0.6% 0.8%

Page 18 / 32

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.

Page 19 / 32

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

1. 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 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.

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 20 / 32

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 21 / 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.

Page 22 / 32

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.

Page 23 / 32

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

15

QUIKView DNA Stain is the new DNA stain to be tested that is manufactured from Carolina company

Page 24 / 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 0.6%

Bacteriological Agar

0.8%

Bacteriological Agar

HindIII Ladder

Base Pair(a)

/ bp Distance from the well/ cm

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 25 / 32

Data points from Chart 3 were plotted using a semi log graph to obtain linear trends, shown in Graph 2 below.

Graph 2: Comparison of Hind III Ladder on standard 0.8% Agarose and 0.6% and 0.8% Bacteriological Agar.

4361

2322

2027

23130

9416

6557

25 30 35 40 45 50 55 60 65

Base

Pair/

bp

Distance from the well/ cm

HindIII Digest on

Different Agar Mediums

0.8% Agarose

0.6% Bacteriological Agar

0.8% Bacteriological Agar

Page 26 / 33

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.

0.6% Bacteriological Agar 0.8% Bacteriological Agar

HindIII 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, 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%

Page 27 / 32

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 28 / 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.

Page 29 / 32

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.

16

A restriction enzyme that digests the DNA at four different sites and produces five fragments of similar

lengths

Page 30 / 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.

Page 31 / 32

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) 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

.

(a) (b) (c) (d)

Page 32 / 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>.