Gel Electrophoresis Lab

Nicole Balsirow, McKenzie Janisz, Madison Linton, Brady Nichols

Block 3 Biotechnology

25 October 2018
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Introduction:

Lambda is a DNA bacteriophage that contains sequences​ of ​genes​ that move around

whilst protected by protein shells​ (Abedon, 2016). This bacteriophage can be cut by restriction

enzymes; specifically, this lab utilizes ​Bam​HI, ​Eco​RI, and ​Hin​dIII enzymes to separate the DNA

into several fragments. The function of restriction enzymes is to “recognize specific sequences of

DNA base pairs and cut, or chemically separate, DNA at that specific arrangement of base pairs”

(Mardigian, 2013). Restriction enzymes, respectfully, are named after the bacterium they are

separated from; ​Eco​RI stemming from ​Escherichia coli​ bacteria, ​Hin​dIII originating from

Haemophilus influenzae​ bacteria, and ​Bam​HI from ​Bacillus amyloliquefaciens​. The use of

restriction enzymes allows scientists to recombine various strands of DNA fragments, known

more commonly as recombinant DNA technology (Mardigian, 2013).

The ​Eco​RI restriction enzyme splits DNA into a ‘sticky end,’ severing the double helix

on both sides between the G and A bases, leaving overhanging single strands of DNA that can

then combine with a new complementary piece (“Restriction Enzyme,” 2009). Similarly to the

​ I enzyme, ​Bam​HI leaves a ‘sticky end’ when separating DNA at two consecutive G bases,
EcoR

much like ​Hin​dIII does when recognizing two consecutive A bases (Mardigian, 2013). As the

three restriction enzymes all cut the lambda DNA bacteriophage in different locations, a process

known as electrophoresis is used to organize the DNA fragments created by the enzymes by size,

with the smaller molecules migrating toward the positive end of an agarose-gel, to distinguish

DNA fragments of different lengths and compare the banding of the DNA molecules with that of

the other enzymes (“What is Gel Electrophoresis?” 2016).

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Methodology:

The lab was conducted across 15 October to 19 October, 2018. The TBE buffer solution

was created by mixing 25mL 20X buffer and 475mL of distilled water in an erlenmeyer flask to

make 500mL of a buffer solution. Of the solution, 60mL was allocated for agarose gel synthesis

in a separate flask. The 0.8% agarose gel was created by adding 0.48g of agarose powder,

weighed in an analytical scale, to the 60mL of the TBE buffer. The agarose solution was

microwaved for 45 seconds on a high setting to dissolve the agarose and allowed to cool until the

flask was able to be held. One drop of Carolina BLU™ stain was then added to the agarose

solution.

To prepare the casting tray, the comb was placed on the black (cathode) end of the tray

to create the DNA wells. Rubber stoppers were utilized on the ends of the tray to ensure that the

gel took the correct form and did not leak. The agarose solution was poured into the tray until a

depth of about 5mm, or 1/3 the height of the comb teeth, was reached. Any bubbles were

relocated to the sides of the tray. The gel was allowed to cool for about 10 minutes. Then the

comb was removed and the gel was placed into a weighing boat with a small amount, just

enough to cover the surface of the gel, of TBE buffer solution for storage in a refrigerator

overnight with parafilm. The remaining flask of TBE

buffer solution was also sealed with parafilm and

placed into the refrigerator.

Figure 1​ was used as a reference for setting up the test tubes. Four

tubes were labeled: ‘B’ for the ​Bam​HI enzyme, ‘E’ for the ​Eco​RI enzyme,
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‘H’ for the ​Hin​dIII enzyme, and ‘-’ for the tube with H​2​O. 4µL of DNA was first added to each

tube using adjustable pipettes with sterilized tips. The 5µL of buffer was

added afterwards with the same method. The enzymes were then each

added to their respective tubes.

The incubation process was set up by floating a plastic test tube

holder in a beaker placed in a sink. The water was kept running at a stable

37°C for approximately two hours. The test tubes were then placed into a

freezer for storage until the next day (​Figure 2​).

Once the material in the test tubes defrosted, 1µL of loading dye was added to each tube

and mixed. The agarose gel was placed back into the casting tray without

the rubber stoppers or comb, lined up with the correct color (black to

black and red to red) and placed into the gel electrophoresis chamber.

Next, TBE buffer was poured into the chamber until it completely

covered the gel and there were no dimples above the wells. To load the

material, the same adjustable pipette was used (with sterile tips) and set to

11µL. The material was expelled over the wells instead of in the wells so

that the gel was not punctured. Once the material was placed into the four

wells, electrophoresis could begin.

The top of the electrophoresis chamber was closed and the power

supply was set to 125 volts. The power supply was turned on and

electrophoresis was run for about 20 minutes (​Figure 3​). When complete,
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the power supply was turned off and the lid was removed from the

chamber. The TBE buffer from the chamber was poured into a waste

container.

The gel was then removed from the chamber and placed in a

weighing boat for staining (​Figure 4​). The gel was covered

completely with Carolina BLU™ and allowed to stain for 20

minutes. When the staining was complete, the stain was poured back

into the container. To observe the bands, the gel was placed on a

light box and a ruler was placed next to the gel for measurement of the

distances that the bands moved.

Post-lab, Microsoft Excel was used for data analysis.

Results:

Table 1​ displays the migration data from each of the three enzymes as well as their

distance-calculated and actual fragment lengths. The calculated fragment lengths were found

using the logarithmic equation from ​Figure 6​. ​Figure 6​ displays the correlation between the

distance the ​Hind​ III fragments migrated and the actual kilo-basepair length of the fragments. The

R​2​-value was 0.9576, indicating that the relationship followed the logarithmic model. ​Figures 7

and 8​ illustrate the correlations between the calculated kilo-basepair length of each fragment and

the distances the fragments migrated for ​Eco​RI and ​BamH

​ I, respectively. The R​2​ of ​Figure 7
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was 1 because there were only two points, however the R​2​ of ​Figure 8​ was 0.9935, indicating

that the data follows the logarithmic model. ​Figure 9​ is the gel on a light box after the process

was complete.

Discussion:

In ​Figure 9​,​ ​the control DNA without restriction enzymes had the least amount of

fragments compared to the samples incubated with ​Bam​HI, ​Eco​RI, and ​Hind​ III enzymes, thus

indicating a reliable control. The lack of restriction enzymes causes the DNA to not cleave into

several bands; as a result, all the DNA is compacted into a single strand, as was expected, and

“[o]btaining expected results with the control organism affirms that (i) the procedure, including

the cell lysis, washing, and endonuclease digestion steps, is working; (ii) the gel and

electrophoretic conditions have been appropriate; and (iii) the conditions of the procedure have

yielded results that are reproducible from run to run within the laboratory and that are consistent

with those obtained by other investigators” (​Tenover et. al​ 1995). In ​Table 1​, there are

differences in the calculated kbp versus the actual given kbp values the instructor manual

provides. The sample incubated with the ​Hind​ III restriction enzyme has 5 observable DNA

bands, but the actual kbp indicates 7 bands each with decreasing kbp. However, in ​Figure 6​, the

amount of bands and the actual amount of base pairs do indicate the negative correlation,

meaning the further away the fragments migrate, the less base pairs that will be in that fragment.

​ I, in ​Table 1​, has only two visible DNA fragments with the calculated fragment length as
EcoR

6.1 kbp and 2.3 kbp. In contrast, the actual DNA has 7 fragments with decreasing size ranging

from 24.8 kbp to 3.5 kbp, showing a discrepancy between the calculated and actual kbp length.
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In ​Table 1​, ​Bam​HI has three observable DNA fragments ranging from 14.2 kbp to 4.9 kbp,

compared to the ideal situation with 7 actual fragments ranging from 16.8 kbp to 5.5 kbp. In

Figure 6​, the relationship between the observed distances and the log of the actual fragment

length fit the logarithmic model based on the R​2​ value of 0.9576. This showed that though there

were differences in the fragment lengths of the observed gel compared to the ideal gel, the

relationship between the distance and fragment length was still accurate.

Compared to the ideal case scenario presented in the

Instructor’s manual for DNA restriction enzyme analysis, ​Figure

5​, the observed bands were not as spread out and separated.

Undertaking the lab, several factors contributed to the outcome;

the gel was ran for 20 minutes with 125V instead of a longer time

with a smaller voltage. As a result, the DNA became denatured

because “Excessively high voltage may result in gel heating and

DNA denaturation… Excessive electrophoresis run times or

voltage may result in migration of small DNA fragments off of

the gel” (“Troubleshooting”). Low voltage conditions, compared

to high-voltage conditions, have a region of intense staining around

molecular weights greater than 1500 kb; this indicates that under

low-voltage conditions, fragments with larger molecular weights can migrate into the gel.

However, the large fragments can create a zone of unresolved fragments (​Ager et al. 1990).

Another factor that influenced the end result of this experiment was the use of the Carolina BLU

stain as opposed to Ethidium Bromide. In a separate experiment it states that “Carolina BLU
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(Carolina Biological Supply Company) worked as well in sodium borate as in TBE buffer when

tested in our lab, however it is three to four times less sensitive than ethidium bromide and

requires at least an hour to stain and destain before samples can be viewed” (​Kristin P. Jenkins,

Barbara Bielec and Kristen P. Jenkins 2006). The differences in the separation of the DNA

fragments can be attributed to many underlying factors.

Conclusion:

To summarize, three different DNA bacteriophages were cut with restriction enzymes

and then electrophoresed to separate the remaining fragments through the medium of gel. The

main purpose of the lab was to determine the distance of which the lambda DNA would migrate

​ I DNA,
in the gel and to separate it into bands. The wells were loaded with samples of ​EcoR

Bam​HI DNA, ​Hind​ III DNA, and a control DNA and electrophoresed. An electrical field applied

across the gel caused the DNA fragments in the samples to move from their origins through the

gel matrix toward the positive electrode. As a result of this experiment, there were significantly

less bands than there were in the ideal gel. However, this was due to the limited amount of time

the lab was conducted in; resulting in separation differences between the outcome and ideal

DNA fragments. Additionally, the lab confirmed that the greater the distance between the origin

and the band, the less amount of base-pairs in that band.

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Acknowledgements

We would like to thank Mr. Adam Sprague for the opportunity to perform the lab and the

instruction in doing so. We would also like to thank MATES OCVTS for the materials and site

used to run the experiments.

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Table 1​ The distances (mm) of the material cleaved by each enzyme and the calculated fragment
length (kbp) compared to the actual fragment basepair length (kbp) provided by the instructor
manual. A single asterisk (*) next to a number means that the pair appears as a single band in the
ideal gel.
​ III
Hind Eco​RI ​ I
BamH

Distance Actual Distance Calculated Actual Distance Calculated Actual

(mm) (kbp) (mm) (kbp) (kbp) (mm) (kbp) (kbp)

7 27.5* 11 6.1 24.8* 8 14.2 16.8

23.1* 16 2.3 21.2* 11 6.1 12.3

9 9.4 7.4 12 4.9 7.2

10 6.5 5.8* *6.8

12 4.4 5.6* *6.5

17 2.3 4.9 *5.6

2.0 3.5 *5.5

Figure 6​ Distances (mm) that the ​Hind​ III material migrated graphed against the logarithm of the
actual kilo-basepair length of material cut by the enzyme (n=5). The line of best fit was used for
calculations in ​Table 1​. The R​2​ value was found to be 0.9576, suggesting that the data points fits
the logarithmic model.
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Figure 7​ Distances (mm) that the ​EcoR​ I material migrated against the calculated base pair length
(kbp) of the fragments (n=2). The R​2​ value is 1 due to there only being two points of data.

Figure 8​ The distances (mm) that the bands of ​BamH ​ I material migrated against the calculated
fragment lengths (kbp) (n=3). The R​2​ value is 0.9935, showing that the relationship between the
factors fits the logarithmic model.
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Figure 9​ The gel on a light box after staining, showing the bands of DNA that migrated during
electrophoresis. B is the well with ​BamH​ I, E is the well with ​EcoR
​ I, H is the well with ​Hin​dIII,
and - is the control well containing DNA and H​2​O. (​Photo credit to Madison Linton​)
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Works Cited

Abedon, S. T. (2016, September 28). Introduction to Bacteriophages. Retrieved October 24,

2018, from http://www.phage-therapy.org/writings/bacteriophages.html

Ager, D., Dewey, W., Gardiner, K., Harvey, W., Johnson, R., & Waldren, C. (1990).

Measurement of Radiation-Induced DNA Double-Strand Breaks by Pulsed-Field Gel

Electrophoresis. ​Radiation Research,​ ​122(​ 2), 181-187. doi:10.2307/3577604

Kristin P. Jenkins, Barbara Bielec, & Kristen P. Jenkins. (2006). Running DNA Mini-Gels in 20

Minutes or Less Using Sodium Boric Acid Buffer. ​The American Biology Teacher,​ ​68(​ 9),

544-546. Retrieved October 24, 2018 from http://www.jstor.org/stable/4452062

Mardigian, R. (n.d.). Restriction Digestion and Analysis of Lambda DNA Kit. ​Biotechnology

Explorer.​ Retrieved October 24, 2018, from

https://www.cpet.ufl.edu/wp-content/uploads/2013/10/Restriction-Digest-and-Analysis-o

f-Lambda-DNA-kit-manual.pdf.

Restriction Enzyme - Action of EcoRI. (n.d.). Retrieved October 24, 2018, from

http://www.accessexcellence.org/RC/VL/GG/restriction.html

Tenover, F. C., Arbeit, R. D., Goering, R. V., Mickelsen, P. A., Murray, B. E., Persing, D. H., &

Swaminathan, B. (1995). Interpreting Chromosomal DNA Restriction Patterns Produced

by Pulsed-Field Gel Electrophoresis: Criteria for Bacterial Strain Typing. ​Journal of

Clinical Microbiology,33​, 2233-2239. Retrieved October 24, 2018, from

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC228385/pdf/332233.pdf.

Troubleshooting Guide for DNA Electrophoresis. (n.d.). ​Protocols and Recommendations for
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DNA Electrophoresis,​447-452. Retrieved October 24, 2018, from

http://res.hmu.edu.iq/Portals/0/Users/Bazhdar/DNA_Troubleshooting.pdf

What is gel electrophoresis? (2016, January 25). Retrieved October 24, 2018, from

https://www.yourgenome.org/facts/what-is-gel-electrophoresis