INTERNATIONAL BACCALAUREATE DIPLOMA PROGRAMME EXTENDED ESSAY
BIOLOGY Is bacteriological agar a viable substitute for agarose in Lambda DNA gel electrophoresis and if yes, what is the optimum gel concentration for it to produce comparable results with agarose as the electrophoresis medium?
Word Count: 3971
by MUHAMMAD KHAIRUL SYAHIR BIN ABD HAKIM 002206-027
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Muhammad Khairul Syahir bin Abd Hakim
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ABSTRACT Restrictive gel electrophoresis is a common means of separating DNA sample into bands depending on the size of the DNA molecules contained in that sample. This method uses restrictive matrix with pores of uniform size dependent on its concentration for the separation. These pores cause greater restriction for bigger molecules and less restriction for smaller molecules, causing them to migrate faster electrophoretically than larger molecules under the influence of electrical field. One common matrix is agarose, a highly purified form of agar. Due to its high purity, agarose is extremely expensive. This research investigates the viability of a much cheaper alternative, bacteriological agar, for restrictive gel electrophoresis and its optimum concentration to produce comparable results to agarose. This research will test the ability of bacteriological agar to resolve single band as well as multiple bands of DNA. These abilities are chosen because they are the most basic application for restrictive gel electrophoresis in educational institutions. Uncut lambda DNA is used as sample for single-band resolution, while the same DNA treated with EcoRI (a restriction enzyme used to cut DNA at specific sites) is used for multiple-band resolution. For the multiple-band resolution, 0.8%, 1.0%, and 1.2% bacteriological agar is used to determine which concentration is optimal in producing comparable results to agarose. The results show that bacteriological agar is able to resolve both single- and multiple-band DNA with 1.2% bacteriological agar producing the closest result compared to 0.8% agarose. 0.8% bacteriological agar is not able to resolve multiple-band DNA, while 1.0% bacteriological agar produces the best resolution. It is found through this research that bacteriological agar is a viable substitute for agarose in restrictive gel electrophoresis under the tested condition. The optimum concentration for bacteriological agar to produce the closest comparative result to agarose is 1.2% for multipleband DNA. (Word count: 298)
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CONTENTS
ABSTRACT ...................................................................................................................................................... 1 CONTENTS ..................................................................................................................................................... 2 1.0
INTRODUCTION ................................................................................................................................. 3
2.0
HYPOTHESIS ...................................................................................................................................... 5
3.0
TESTING THE ABILITY TO RESOLVE SINGLE BAND OF DNA ............................................................... 7
3.1
Modification to the gel slab .......................................................................................................... 7
3.2
Devised method ............................................................................................................................ 8
3.2
Data Collection ............................................................................................................................ 11
3.3
Data Analysis ............................................................................................................................... 12
4.0 TESTING THE ABILITY TO RESOLVE / SEPARATE MULTIPLE BANDS OF DNA AFTER TREATED WITH RESTRICTION ENZYME................................................................................................................................. 14 4.1
Devised method .......................................................................................................................... 14
4.2
Data Collection ............................................................................................................................ 17
4.3
Data Analysis ............................................................................................................................... 18
5.0
EVALUATION ................................................................................................................................... 20
6.0
CONCLUSION ................................................................................................................................... 23
APPENDIX .................................................................................................................................................... 24 BIBLIOGRAPHY ............................................................................................................................................ 26
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INTRODUCTION
Gel electrophoresis is a common method of separating deoxyribonucleic acid (DNA) of different sizes (different number of base pairs) into visible bands for further analysis. This method is included in the Biology subject of the International Baccalaureate Diploma Programme (IBDP). However, practical skills are as important as mastering the knowledge and theories in the subject, and theoretical knowledge of an experiment does not guarantee the practical capability of performing that experiment at all. These practical skills must be acquired through actual practice. Despite that, students rarely have the chance to perform DNA gel electrophoresis themselves in many institutions for pre-university educations. It invokes my curiosity as to why many institutions do not include this particular practical experience into the course. The main reason for this exclusion, as I later learned is because of the costs for such experiment. A typical gel electrophoresis apparatus costs around USD1000.00. 100g of Standard analytical grade agarose (the gel used in the gel electrophoresis) can cost anywhere from USD89.60 to USD297.70. Other necessary equipments such as micropipette and microcentrifuge will also incur significant costs, as do the reagents used to produce necessary buffers, loading dyes, and staining solution. While the equipment costs to run such experiment can be considered as one-time costs, the materials needed to run the experiment that cannot be reused are not. The major cost for each run is incurred by agarose. It occurs to me then that since agarose is just a form of ultra-purified agar derived from seaweed, finding a cheaper substitute for agarose could therefore enable educational institutions to provide the facility to perform DNA gel electrophoresis. Initially, I planned to include a wide variety of commercial agar of various brands to identify possible substitute for agarose. However, after several experiments, I found out that the
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staining process left the agar coloured. This leads to a modification in the research question. Bacteriological agar is chosen as a primary candidate as it displayed the best result among all the agars tested, and still costs relatively much cheaper compared to agarose. Therefore, my precise research topic is:
Is bacteriological agar a viable substitute for agarose in Lambda DNA gel electrophoresis and if yes, what is the optimum gel concentration for it to produce comparable results with agarose as the electrophoresis medium?
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HYPOTHESIS
Restrictive gel electrophoresis works by loading negatively charged molecules like DNA in wells formed in a restrictive matrix such as agarose gel which is then submerged in a buffer solution. Electrical field is applied to the buffer solution, causing the charged molecules to migrate in the direction of the opposite charge. The presence of phosphate group in DNA molecule causes it to be negatively charged, hence causing it to migrate in the direction of the positive charge electrode. Agarose, a highly-purified agar made from red seaweed1, is usually used in gel electrophoresis. The gel formed will have pores with roughly
a
uniform
size
which
depends on the concentration of agarose, with bigger pore size for lower agarose concentration and vice versa. As DNA molecules Figure 1: The phosphate molecules in the backbone of the DNA double helix structure give DNA its negative charge
migrate, these pores restrict their movements. Their rate of migration
of the DNA molecules of the same size is the same because of the uniform pore size. Smaller DNA molecules will migrate with less restriction, hence their rate of migration is higher than bigger DNA molecules, and vice versa. This property forms the basis of the gel electrophoresis method.
1
(Westermeier)
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Following the principle described, I hypothesise that any matrix that has regular pore size should be able to produce the same result as agarose. Therefore, based on this hypothesis, I have chosen to test the viability of bacteriological agar to be used as a cheaper substitute for agarose in restrictive gel electrophoresis. Since agarose is a form of highly-purified agar (agar nonetheless), therefore I believe that bacteriological agar should be able to replace agarose in restrictive gel electrophoresis, as this agar should also have the same property of uniform pore size as would any other agar. Bacteriological agar is chosen instead of other commercial brands agar because it contains minimal amount of impurities.
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3.0
TESTING THE ABILITY TO RESOLVE SINGLE BAND OF DNA
3.1
Modification to the gel slab
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Since this research involves testing and comparing the results of multiple agars both of different types and different concentrations, the available gel slab in the school which allows for only 2 agars to be tested at once will require many repetitions of the gel electrophoresis process. This will greatly increase the time needed for the research and will far exceed the time allocated by IBO. Apart from that, comparison of the results is best done when all the agars are tested at once to ensure that all the other constant variables are actually constant. For these reasons, I decided to make some modifications to the available gel slab. A sheet of acrylic plastic is cut into small pieces to make as dividers in the gel slab. The precise size of the divider is measured beforehand and sharp cutter is used to cut the acrylic. The actual process took a lot of effort and satisfactory result is hard to achieve. After the dividers are cut, they are attached to the original gel slab by using chloroform. The resulting gel slab is now able to contain four different agars or different concentration of agars, twice the original capacity.
Dividers cut from acrylic plastic, attached using chloroform
Original gel slab with 2 gel compartments
Modified gel slab with 4 gel compartments
Figure 2: Modification of the gel slab
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Devised method
To test the ability of bacteriological agar and commercial agars to resolve single band of DNA by using the restrictive gel electrophoresis method, DNA sample which contains DNA molecules of the same size will be used. According to the hypothesis, the bacteriological agar and commercial agars should be able to resolve these uniform-sized molecules into just a single band. For this purpose, the Lambda (λ) DNA2 with the size of 48,502 base pairs3 (bp) will be used.
Gel preparation 1. 0.4g of bacteriological agar is weighed and added into 50ml of 1x TBE4 buffer. 2. The mixture is heated using microwave oven and stopped when it starts to boil, and swirled to aid the dissolving process. 3. Step 2 is repeated until the mixture forms a clear solution and then let to cool to around 50° C. This will make up a 0.8% bacteriological agar solution. 4. Agar solution is then poured into a gel slab and a well comb is placed at one end of the slab. The agar is left to solidify. 5. Steps 1 to 4 are repeated for agarose and two brands of commercial agars.
DNA sample preparation 1. A mixture of 3μl λ DNA, 14μl distilled water and 3μl loading dye with bromophenol blue is prepared in a microcentrifuge and loaded into the first well using a micropipette. 2
Lambda DNA is a common substrate used in restriction enzyme activity assay. It is a linear, double-stranded DNA obtained from the lambda phage virus. The Lambda DNA used in this research is obtained through the school from Fermentas International Inc. 3 (Fermentas International Inc) 4 Recipe for all solutions used in this experiment is provided in Appendix
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2. Step 1 is repeated but using 6μl λ DNA, 11μl distilled water and 3μl loading solution without bromophenol blue, which is then loaded into the third well. 3. Steps 1 to 2 are repeated using agarose to be used as control, and afterwards using the commercial agars. All gels are then transferred onto the gel slab, which is then put inside a gel electrophoresis apparatus and is submerged in a homogenous 1x TBE buffer to ensure a constant pH throughout the electrophoresis process. 4. Electrical current with 50V voltage is applied and the apparatus is left until the bromophenol blue marker (migration rate equal to approximately 300bp5) reaches near the end of the gel slab at the other side.
Staining 1. All gels are transferred into another clean container, into which methylene blue solution is poured to stain the DNA. Methylene blue is chosen as it is generally harmless and stains the DNA molecules quite well as it is a positively-charged organic dye which will attach to the negatively-charged DNA molecules and will stay there when the agars are destained using distilled water. Ethidium bromide (EtBr) is not used due to safety reasons as EtBr is a potent mutagen and known carcinogen. 2. The staining process is done for 15 minutes, after which the methylene blue is removed and the agars are destained using distilled water. 3. Steps 1 to 3 are repeated until a good contrast between the DNA band and the agar is achieved.
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(American Phytopathological Society)
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4. The results are then photographed.
Photographing method Since the experiment uses methylene blue solution to stain the DNA instead of EtBr, therefore the visibility of the bands after staining is less. Getting the visible bands into the photographs captured is quite a challenge, as normal method will render the bands not visible at all. To overcome this problem, a white background (foam board) and a 100W light bulb is used to produce adequate contrast for the bands to be visible. The gels are then photographed under this condition.
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Data Collection
The following result is obtained.
0.8% agarose
0.8% bacteriological agar
0.8% “Pearl Mermaid” agar
0.8% “Nona” agar
Commercial agars
Figure 3: The results of gel electrophoresis for agarose, bacteriological agar, and commercial agars for single-band resolution. The white boxes indicate the position of visible bands.
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Data Analysis
From the results obtained, all agars tested including the commercial brands show clear bands of the DNA after staining and destaining, and the position of the band is around the same distance from the wells with those in agarose. This indicates that all the agars do have the ability to resolve single-band DNA. It appears that this early result supports the hypothesis. All the agars including agarose show some smearing of the DNA band produced. This may be due to the large amount of DNA used in this experiment (with the purpose of ensuring visibility of band after staining). Though the commercial agars resolve the single-band DNA pretty well, the staining process left the agar heavily coloured, as shown in Figure 3 below.
Figure 4: Agarose, bacteriological agar, and commercial agar brands Pearl Mermaid and Nona after staining with methylene blue and destaining with distilled water.
Agarose remains uncolourised after staining, while bacteriological agar is minimally colourised. “Pearl Mermaid” and “Nona” agars, however, are heavily colourised and it is much harder to see the bands in the two agars without image manipulation. Methylene blue staining solution might 12
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have bind to the charged impurities particles in the agars and produce the background colour, which significantly affects the visibility of the bands produced. This result prompted me to narrow down the research only to bacteriological agar as a possible agarose substitute, and made modifications to the research question.
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4.0 TESTING THE ABILITY TO RESOLVE / SEPARATE MULTIPLE BANDS OF DNA AFTER TREATED WITH RESTRICTION ENZYME
4.1
Devised method
Since DNA molecules of different sizes migrate through the gel matrix in different rate of migration, therefore the bacteriological agar should be able to resolve these molecules into multiple bands, with smallest DNA molecules making up the farthest band and biggest DNA molecules making up the nearest band. To test this ability, we will also be using λ DNA but this time treated with EcoRI restriction enzyme that will cut the DNA molecules at 5 specific sites, producing 6 fragments. For this experiment, two DNA samples will be prepared. The first sample will contain uncut DNA (no restriction enzyme introduced) which will serve as control, and the second sample will contain DNA molecules that have been treated with EcoRI. Using these two samples will also allow me to determine if all the DNA molecules in the second sample has been digested by the restriction enzyme.
Preparing uncut DNA sample 1. 12μl of distilled water, 5μl of λ DNA, and 3μl of loading solution without bromophenol blue is added into a clean microcentrifuge. 2. This mixture is mixed gently and spinned for several seconds using a centrifuge.
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Preparing cut DNA 1. 10μl of distilled water, followed by 2μl of 10x EcoRI buffer solution, 5μl of λ DNA, and lastly 3μl of EcoRI enzyme is added into a clean microcentrifuge in order. The overall volume of the mixture (20μl) will dilute the 10x EcoRI buffer solution into 1x buffer, producing the optimum buffered solution for the enzyme’s digestion activity. Steps are done in specified sequence to avoid premature enzyme activity. 2. The whole mixture is mixed gently and spinned for several seconds using centrifuge. 3. The mixture is then incubated in a 39.5°C6 water bath for 2 hours to provide optimum condition for the enzyme activity and ensure that digestion is complete. 4. Steps 1 to 3 are repeated for samples to be used with agarose as control. Loading dye with bromophenol blue is used instead of the loading solution without bromophenol blue to serve as marker for the electrophoresis process.
Gel preparation 1. 0.8% bacteriological agar is prepared by dissolving 0.4g of bacteriological agar powder into 50ml of 1x TBE buffer. 2. Step 1 is repeated for agarose.
Running the sample 1. The uncut DNA samples are loaded into the second well of each bacteriological agar gel and agarose while the cut DNA samples are loaded into the fourth well.
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The optimum temperature for the enzyme activity is actually 37°C, but 39.5°C water bath is used to compensate for heat loss during transfer from the water bath to the solution inside the microcentrifuge. This condition is tested with distilled water where the temperature of the distilled water inside the microcentrifuge is measured by using an electronic temperature probe before the actual DNA-enzyme mixture is incubated.
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2. 50V current is applied and the apparatus is left until the bromophenol blue marker reaches the other end of the agar. 3. The gels are then transferred into a clean container and destained using distilled water. 4. The gels are photographed under the same condition as the single-band experiment.
The same experiment is repeated by using 1.0% and 1.2% bacteriological agar prepared by dissolving 0.5g and 0.6g of bacteriological agar powder respectively.
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Data Collection
Agar Concentration (%) Results
Agarose 0.8%
Uncut DNA
0.8%
Uncut DNA Cut DNA
Cut DNA not visible
Bacteriological Agar 1.0%
1.2%
Uncut DNA
Uncut DNA Cut DNA
Cut DNA
Control
Table 1: The results of the experiments which shows agarose and bacteriological agar's ability to resolve multiple bands of DNA at different concentrations. The white arrows indicate the position of visible bands.
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4.3
Data Analysis
From the side-by-side comparison of the agars used in gel electrophoresis, we can see that 1.0% and 1.2% bacteriological agar were able to resolve DNA with multiple sizes and produce multiple bands as a result, comparable with those of 0.8% agarose. 0.8% bacteriological agar did not produce any bands for DNA sample treated with restriction enzyme EcoRI. All four agars including the 0.8% bacteriological agar do, however, produce a single band for the uncut DNA sample. It is noted that both 1.0% and 1.2% bacteriological agar yield only four visible bands, and so does 0.8% agarose, even though treatment with EcoRI produces 6 fragments of different sizes. This is likely because of one pair of the DNA fragments have very little difference in size as shown in Table 2 below (fragments 3 & 4), hence these two fragments will only show up as a single, combined band. Fragment 6 might not be visible as a band due to its small size (only 3530bp) that not enough bromophenol blue is attached to it to make it visible. Comparison with external sources confirmed that only 4 bands are visible7. Fragment 1 2 3 4 5 6
Size (base pair) 21226 7421 5804 5643 4878 3530
Table 2: Fragment sizes of EcoRI-treated 位 DNA
There is no visible band in lane 4 at the same site where the single band is visible on lane 2 for 0.8% agarose, 1.0% bacteriological agar and 1.2% bacteriological agar, indicating that the DNA samples are completely digested by EcoRI. 1.0% bacteriological agar shows the best band resolution, with clear separation between each band of the cut DNA. 0.8% agarose and 1.2% bacteriological agar show identical distinct separation between the bands of the cut DNA, but separated more closely. This might be due to the relatively less gel strength of the bacteriological agar compared to agarose. Bacteriological 7
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agar has gel strength8 of 500g/cm3 while agarose has gel strength of 700g/cm3. Therefore, it could be that the pore size in 1.2% bacteriological agar is the same with 0.8% agarose that leads to the identical resolution/separation. 1.0% bacteriological agar, on the other hand, has a greater pore size than both 0.8% agarose and 1.2% bacteriological agar, hence it shows greater separation of bands. This is summarised in Table 3 below: Pore size comparison 0.8% bacteriological agar > 1.0% bacteriological agar > 1.2% bacteriological agar = 0.8% agarose Table 3: Pore size comparison between the agars and their respective concentrations used in the experiment
This result also confirms our hypothesis that greater gel concentration will yield smaller pore size of the agar, which will cause DNA molecules to migrate more slowly and hence, separated more closely.
The 0.8% bacteriological agar was not able to resolve DNA with multiple sizes probably because of insufficient gel strength. The low concentration may cause a significant part of the agar to be still in liquid form and therefore, is not able to resolve the multiple-sized DNA molecules. It is observed that during gel preparation, the gel did not solidify properly even after placing it under the fan for more than 20 minutes.
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By reference to the labels of the gels bottles
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EVALUATION
The experiments performed showed that bacteriological agar is able to resolve both uncut and cut DNA samples (using EcoRI restriction enzyme), producing a single band and multiple distinct bands respectively. Under the tested condition, bacteriological agar did in fact produce comparable results to that of agarose. During gel preparation, gel slabs are left under the fan for about 20 minutes after liquid agar is poured to allow the agars to solidify, which may cause agar dehydration. However, this is not likely to affect the result as the agar will be submerged in 1x TBE buffer solution during the electrophoresis process. This buffer solution is used to prepare the agar, so any dehydration during the solidification process will likely be eliminated as the agar is rehydrated by the buffer solution. Throughout the research, the experiment is done by using TBE buffer9 prepared from the same batch. The pH may change slightly over time or during dilution, but this is not likely to give much effect on the experiments as the main purpose of using the buffer is to keep the pH of the agar and the sample constant so that the net charge on the DNA sample will always be negative. As long as the buffer is alkaline with pH around 8.2, this objective is achieved. During the destaining process, the bacteriological agar takes a longer time and has to be destained separately from agarose. There was also noticeable discolouration at the opposite end of the well, which might be due to charged particles of impurities in the bacteriological agar that migrates in the same direction with the DNA samples. These impurities might bind with methylene blue and causes the discolouration. Agarose did not display this discolouration, which could be explained by the absence of impurities as agarose is a highly pure form of agar.
Uncertainties and limitations One possible source of uncertainty is the concentration of the agar prepared. Initially, the mass of agarose and bacteriological agar powder is mixed with an exact volume of water to produce accurate concentration of the resulting solution. However, during heating some water is 9
See Appendix for reagents and methods used in preparing the TBE buffer
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lost as water vapour and this decreases the volume of water. As a result, the concentration of the agar solution may not be accurate. All DNA samples used in this experiment is taken from the same batch. There may have been contamination of the sample or some of the DNA might have been denatured. The fact that the school refrigerator can only reach -4°C which is not adequate for DNA storage adds to this possibility. In this research, agar strength is not given much emphasis, only the pore size of the agar. Agar strength is an important factor in gel electrophoresis of DNA, as it determines whether the DNA sample can be extracted from agar after electrophoresis for further tests or experiments. This has not been included in the research. The viability as a substitute is, however, based solely on the agar’s ability to resolve single-size and multiple-sizes fragments of DNA samples to produce single and multiple bands respectively. 1.2% concentration of the bacteriological agar is chosen as the optimum concentration specific to 0.8% agarose to produce the closest comparative results, without taking into account whether the resolution is at its best or not. There is no external repetition of the experiments, therefore the results may or may not be reproducible, especially so under different conditions. This research tries to make a generalisation based on a fixed condition with little variability. As such, any external repetition of the experiment will give more weight and insights on the derived conclusions.
Ways of improvements The uncertainties in gel concentration can be minimised by marking the initial water level inside the conical flask before heating. After the heating process, distilled water can be added until the water level reaches the marker to ensure accurate concentration of the agar solution produced. Contamination of DNA sample can be minimised or avoided by minimising the number of times DNA sample is taken from the master batch. This can be done by doing as much
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experiment as possible at once while still maintaining proper control and observing proper scientific practices. A collaboration effort with other IB school can be done for attempts of reproducing the results found in this experiment. This will provide external independent results that will give additional weighs and insights on the conclusion found in this experiment.
Further Research Due to time constraint, this research only focuses on the ability of bacteriological agar to resolve single band and multiple bands of DNA under a fixed situation. Only 1 type of buffer is used and the experiments are performed using only λ DNA. This gives rise to the possibilities of further studies to investigate the bacteriological agar’s electrophoretic ability under different condition, such as using different buffer and for different purposes, such as resolving DNA of much smaller sizes (e.g. less than 500bp). It is also possible to test for different concentrations of the agar solution and actually quantify the resolution by using a ladder marker. One other possible study is to test for the optimum voltage for the electrophoresis process, since voltage affects the rate of migration and thus, the separation/resolution of the bands. However, since it is not possible to conduct these extensive studies within the available 40 hours time frame, this research focuses solely on the basic ability of the bacteriological agar to resolve DNA samples. Further researches may need to be done before educational institutions can adopt bacteriological agar as a substitute for agarose with adequate confidence. Apart from that, this research uses λ DNA which is very pure and expensive. As an alternative, schools or colleges may extract DNA from everyday materials and substances, such as onions and broccoli which is much cheaper and can be performed relatively easily using kitchen materials. This is a more practical, economical and user-friendly source of DNA sample. However, the extracted DNA may contain impurities that can affect the result or cause the gel electrophoresis method to fail entirely. Further research is necessary to explore this possibility.
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CONCLUSION
It is found through this research that bacteriological agar is a viable substitute for agarose in restrictive gel electrophoresis, and the optimum concentration to produce the closest comparative results with agarose is 1.2% for 0.8% agarose. The results also show that the pore size of the gel matrix is dependent on the gel concentration, with higher gel concentration producing smaller pore size and lower gel concentration producing bigger pore size, which is indicated by the rate of migration of DNA molecules which is faster in lower gel concentration and slower in higher gel concentration. This confirms the hypothesis of this experiment. However, the condition under which this research is done is strictly fixed. This brings about new questions, one of which is what effect any changes in the variables might have, such as using onion DNA instead of 位 DNA. Apart from that, it is also unknown whether the DNA sample can be extracted again from the bacteriological agar for further analysis of the same sample.
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APPENDIX Preparation of 10X TBE buffer Reagents: 1. 2. 3. 4.
108g tris base 55g boric acid 20ml 0.5M EDTA (pH 8.0) Distilled water
Methods: All reagents are dissolved in water with volume of slightly less than 1.0 litre. If white clumps form, the solution is heated slowly until all the white clumps dissolve. After all reagents have been dissolved, i.e. no visible precipitate, distilled water is added until the volume reaches 1.0 litre. Preparation of working 1X TBE buffer 100mL of 10X TBE buffer is added to 900mL of distilled water. Preparation of 0.025% methylene blue staining solution Reagents: 1. Methylene blue trihidrate powder 2. Distilled water Methods: 1g of methylene blue trihydrate powder is dissolved into 100ml of distilled water. 25ml of the resulting solution is added into 975ml of distilled water to make up 1 litre of 0.025% methylene blue staining solution.
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Preparation of 6X loading dye/solution Reagents: 1. 2% bromophenol blue solution 2. 50% glycerol solution 3. Distilled water Methods: Preparation of 2% bromophenol blue solution and 50% glycerol 0.2g of bromophenol blue powder is dissolved in 10ml of distilled water to make up 10ml of 2% bromophenol blue solution. 10ml of glycerol is added into 10ml of distilled water to make up 20ml of 50% glycerol. Preparation of the 6X loading dye/solution 6.0ml of 50% glycerol is mixed with 1.0ml of 2% bromophenol blue solution and 3.0ml of distilled water to make up 10ml of 6X loading dye. For loading solution without bromophenol blue, 6.0ml of 50% glycerol is added into 4.0ml of distilled water to make up 10ml of loading solution.
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BIBLIOGRAPHY American Phytopathological Society. APSnet Education Center - K-12 Lessons and Laboratories - Classroom Activities in Plant Biotechnology - Activity 3. 28 February 2008. 19 August 2008 <http://www.apsnet.org/education/k12plantpathways/TeachersGuide/Activities/PlantBiotechnology/activity3.htm>. Anderson, Nadja. Restriction Enzyme Analysis of DNA - Teacher Guide. 21 September 2004. 22 September 2008 <http://biotech.biology.arizona.edu/labs/DNA_analysis_RE_teacher.html>. Bollag, Daniel M., Michael D. Rozycki and Stuart J. Edelstein. Protein Methods. 2nd Edition. New York: Wiley-Liss, 1996. Delgado, ﾃ］gel V., ed. Interfacial Electrokinetics and Electrophoresis. New York: Marcel Dekker, Inc., 2002. Fermentas International Inc. Molecular cloning, vectors: Lambda DNA. June 2008. 18 August 2008 <http://www.fermentas.com/catalog/nucleicacids/dna_sd0011.htm>. Green, John and Sadru Damji. Chemistry - For Use with the International Baccalaureate Diploma Programme. Victoria: IBID Press, 2001. Hames, B. D., ed. Gel Electrophoresis of Proteins: A Practical Approach. 3rd ed. Oxford; New York: Oxford University Press, 1998. Holtzhauer, Martin. Basic Methods for the Biochemical Lab. 1st English ed. Berlin: Springer, 2006. Westermeier, Reiner. Electrophoresis in Practice: A Guide to Methods and Applications of DNA and Protein Separations. Weinhem: Wiley-VCH, 2005. Yakushevich, Ludmila V. Nonlinear Physics of DNA. 2nd, rev. ed. Weinheim: Wiley-VCH, 2004. Zamora, Antonio. Proteins, Amino Acids, Peptides, and Polypeptides - Chemical Structure. 19 November 2007. 13 March 2008 <http://www.scientificpsychic.com/fitness/aminoacids1.html>.
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