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DNA Restriction Analysis Lab

Michael Hudak, Ava Kennedy, Joseph Kane, Kyle Lopes, Erin Geoghegan, Lucas Monchinski

Marine Academy of Technology and Environmental Science

Biotechnology Block 3

Mr. Sprague

November 13, 2020

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Introduction: EG

Discovered in bacteria and other prokaryotes, restriction enzymes (restriction

endonucleases) are DNA-cutting enzymes that recognize specific sites in DNA, known as

restriction sites. Each one of these restriction enzymes only recognizes just one or a few

restriction sites, making a double-stranded cut in the DNA molecule when it finds its target

sequence (Smith, H., & Nathans, J. (2007)). These cuts are usually predictable. It has been

theorized that restriction enzymes emerged in bacteria as an immune or defense mechanism,

allowing them to cut through foreign and potentially harmful genetic material, such as those

from viral bacteriophage, before they can harm the cell. This process of cutting DNA is referred

to as restriction digestion. Restriction digestion comes into effect when the bacteria in a cell that

is invaded by viral DNA produces methylase and restriction enzymes are able to recognize the

native and foreign units in the cell. The enzymes must experience a palindromic sequence of

nucleotides in bacterial DNA in order to recognize DNA and cleaves at restriction sites in order

to restrict the virus. The restriction enzymes used in this experiment are BamHI, EcoRI, and

HindIII. The BamHI restriction enzyme is able to recognize short DNA sequences, as well as

cleaving them at a specific location. The EcoRI restriction enzyme is able to cleave DNA at

specific sites as well, along with isolation from E. coli. The HindIII restriction enzyme is a

DNase that cleaves DNA when Mg²⁺ is present using hydrolysis, along with isolation from

Haemophilus influenzae. The original discovery of restriction enzymes allowed for scientists to

perform and study genetic engineering. The restriction enzymes are used to cut out sectors of

DNA in comparison to the desired gene and integrate new genes into the cell (Griffiths, A.

(2000, January 01)). In the lab, restriction enzymes were observed and used in order to splice
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DNA. The restriction enzymes were able to be analyzed after being run through gel

electrophoresis, which demonstrates where each enzyme spliced the DNA.

Methodology: AK

A 300 mL 1x solution of tris-borate-EDTA buffer and distilled water was prepared with

285 mL of distilled water and 15 mL of a 20x dilution of the

buffer in an erlenmeyer flask. Forty mL of this solution was

used to create an agarose solution that is used for the gel

plate in the electrophoresis chamber. A 28% agarose

solution was prepared using .32 grams of agarose and 40

mL of the buffer solution. The solution was heated for about

45 seconds and swirled until the agarose was completely

dissolved. The casting tray was prepared with rubber

stoppers on both ends and the comb placed near one of the

ends, as can be seen in Figure 4. Two drops of CarolinaBLU

Gel and Buffer Stain were added to the flask with the 40 mL agarose solution which was left to

partially cool. The solution was then poured into the casting tray until a depth of approximately 6

mL was reached, and was then left to solidify for 25 minutes. Once the agarose gel had

solidified, shown in Figure 1, it was removed from the tray and placed in a weigh boat

overnight.

Restriction Digests were prepared using the following restriction enzymes:

BamHI, EcoRI, HindIII, as well as a digest prepared with no enzyme as a control with uncut

DNA. A 1.5 mL tube was used for each enzyme and a new tip was used on the micropipette for
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each reagent to prevent cross contamination. Each tube recieved 4 μL of DNA, 5 μL of buffer,

and 1 μL of the appropriate enzyme, measured using a micropipette. The control received 1 μL

of distilled water in place of the enzyme. Each tube was individually labeled with its

corresponding enzyme. The solutions were flicked and swirled to ensure that all of the materials

were mixed thoroughly and the enzyme came into contact with both the DNA and the buffer. The

four tubes containing the restriction enzymes were placed into a running water bath at a constant

temperature of 37 degrees celsius, shown in Figures 5 and 6. The tubes were agitated in the water

bath for approximately 35 minutes before being removed and frozen overnight.

The gel electrophoresis was performed the following day after the restriction digests and

gel were prepared. The gel was placed back into the casting tray in the electrophoresis chamber

with the wells on the black end so that the banding would occur moving towards the red side.

The remaining 260 mL of the tris-borate-EDTA buffer and distilled water solution was poured

into the electrophoresis chamber so that it filled the chamber on both sides and covered the top of

the plate with a thin layer of solution as well as completely filling the wells. The DNA samples

were removed from the freezer and warmed. One μL of each restriction enzyme solution was

placed into the gel chamber wells using the micropipette. The samples were placed into the wells

at the surface and were allowed to sink down to the bottom of the wells in the gel casting. The

chamber was connected to the power supply from anode to anode and cathode to cathode and set

to a voltage of 110 for 35 minutes. After the electrophoresis was complete, the gel plate was

removed from the chamber using gloves and the excess buffer solution was poured back into an

erlenmeyer flask. The gel plate was transferred to a weigh boat and covered with CarolinaBLU

final stain, visible in Figure 2. The solution was agitated over the gel for five minutes and then

poured back into the container.

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Data: AK/ KL

The collected data differed greatly from the expected data and results of the ideal

experiment, as can be clearly seen in provided Figures 2 and 3.

Table 1: Distance from well and the thickness of each band created from the corresponding

restriction enzyme. Used ideal data since the experiment did not yield any clear results.

Restriction Enzyme Distance From Well (mm) Thickness (mm)

EcoRI N/A N/A

BamHI 5 1

HindIII 5 2

(-) no enzyme 5 3
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Figure 1: Agarose solution mold after Figure 2: Agarose mold post-electrophoresis

solidifying for 10 minutes and being removed submerged in CarolinaBLU final stain.

from the casting tray. Image taken prior to Banding should be visible at this point.

electrophoresis.

Figure 3: Ideal Restriction Digest of lambda Figure 4: The agarose solution after being

DNA poured into the casting tray. The casting tray

was prepared with rubber stoppers on both

ends. The gel was left to solidify without

movement for at least 10 minutes. The comb

is visible on the right side of the tray creating

the wells in which the restriction digests

would later be inserted in.

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Figure 5: Image of the 4 restriction digests Figure 6: The water temperature was

after being prepared with appropriate measured constantly with a thermometer as

enzymes. The digests were submerged and water was consistently flowing around the

agitated in 37° celsius water for 35 minutes. restriction digests.

Results: JK

No meaningful data were collected over the course of the experiment. The intent of the

lab was to produce bands in the gel corresponding to the enzymes used, which could then be

analyzed. An example of such a result can be seen in Figure 3. The specifics of the bands of an

ideal result can be found in Table 1. The distance from the top of the well to where the bands

formed as electrophoresis went on remained consistent at 5 mm, regardless of the particular

enzyme, while the width of the bands varied. Figure 2 shows the results of the actual conducted
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experiment. No data on the results were collected, as no bands were visible at the conclusion of

the experiment.

Discussion: MH / AK

The restriction enzyme analysis was conducted with as much skill and precision as

resources allowed. However, the results of the analysis were less than ideal. The experiment

failed to produce any meaningful results as no bands of DNA appeared anywhere on the gel. In

an image of the ideal gel, there were five distinct bands of DNA cut by the BamHI and EcoRI

enzymes. There were four bands of DNA cut by the HindIII enzyme (Carolina Biological

Supply, 1995). The lack of bands on the gel after electrophoresis is likely the cause of some

experimental error. First, the bands may have appeared as underloaded. This would indicate that

there was too little DNA in the restriction digests which is likely to have been caused by

inaccurate measurements with the micropipette. There are multiple situations in which

measurements could have been inaccurate, as the lab was conducted on multiple days and the

materials were handled by many people. Incorrect labeling of the digests or gel plate is also

likely to have contributed to the lack of results. It is also possible that the post-electrophoresis

gel may not have been examined properly. The lab manual calls for examination of the gel using

a projector or a light box, neither of which were available for this experiment. The abundance of

variables and a combination of these factors is likely to have contributed to the absence of bands

on the completed electrophoresis gel.

The ideal gel digest shows several distinct bands of DNA for each restriction enzyme,

and only one band for the controlled variable of uncut DNA with no restriction enzyme. To

achieve an ideal gel plate, the DNA must have been properly cut by the restriction enzymes and
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dyed sufficiently using the CarolinaBLU stain provided. By using different enzymes, the ideal

gel digest shows the results of where the different restriction enzymes splice the DNA compared

to uncut DNA prepared with distilled water rather than an enzyme. Through the process of

electrophoresis, the cut strands of DNA are moved across the gel towards the positive electrode

by an electrical field. Larger cut fragments of DNA do not move as far as smaller fragments

(Libretexts, 2020). Thus, the experiment shows the differences among the enzymes including the

number of fragments they create and the relative size of those fragments. However, sometimes

DNA fragments of similar size will move the same distance through the gel, so they are

indistinguishable from each other. As a result, a doublet forms which can be seen as a brighter

and thicker band (GoldiesRoom, n.d.). In order to separate these similar fragments, a larger gel

plate can be utilized as well as extending the time for electrophoresing and adjustment to the

voltage. The larger gel plate will provide more space for the fragments to separate and changes to

time and voltage will yield different results in terms of the spacing of the bands. Variables such

as these contribute greatly to the results of the electrophoresis and adjustments will completely

change the resulting gel stain. If the experiment were to be conducted again, these adjustments

would be taken in consideration, and a more clear analysis is likely to result.

Conclusion: LM

In conclusion, the key to getting good results is ensuring that the procedure is followed

flawlessly and that the electrophoresis is run for the correct amount of time. The results gathered

did not find any bands on the experimental gel, likely a combination of experimental error and

observational error. However, if this experiment were to be repeated, it is likely that these

oversights and mistakes would be rectified.

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Comparing our stained gel (fig. 2) to the ideal gel (fig. 3), we are unable to account for

any fragments of lambda DNA in any lane as they are unidentifiable. However, experimental and

observational error account for the differences in band separation and intensity. In general,

differences in band intensity and separation are related to properly forming wells, removing all

bubbles, making sure it is set before removing the comb, properly micropipetting the contents of

the restriction digests into the wells, being careful not to puncture the wells, and various other

variables.

By changing the concentration of agarose the gel, you can resolve doublet fragments.

Tighter gel matrices separate smaller DNA fragments more effectively, while looser gel matrices

separate larger fragments more effectively. Casting a longer gel or extending the DNA

electrophoresis time could also resolve doublet fragments.

Acknowledgements: MH

This research would not have been possible without the help of Michael Hudak, Joseph

Kane, Ava Kennedy, Erin Geoghegan, Kyle Lopes, and Lucas Monchinski. Special thanks should

be given to Mr. Adam Sprague for his guidance. Finally, gratitude goes out to the Marine

Academy of Technology and Environmental Science for granting the use of its science

laboratories.
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References KL, MH

Carolina Biological Supply Company. (1995). DNA Restriction Analysis Kit. Retrieved

November 12, 2020, from

[Link]

Griffiths, A. (2000, January 01). Making recombinant DNA. Retrieved November 12, 2020,

from [Link]

GoldiesRoom. (n.d.). Lab 14 - DNA Restriction Analysis. Retrieved November 12, 2020, from

[Link]

4%20-%20DNA%20Restriction%20Analysis%[Link]

Harris, K. (2019, March 02). How Are Restriction Enzymes Used? Retrieved November 12,

2020, from [Link]

Libretexts. (2020, September 07). 52: DNA Restriction and Electrophoresis. Retrieved

November 12, 2020, from

[Link]

biology_Labs/Microbiology_Labs_I/52:_DNA_Restriction_and_Electrophoresis

Restriction analysis. (n.d.) Collins Dictionary of Biology, 3rd ed.. (2005). Retrieved November

12 2020 from [Link]

Smith, H., & Nathans, J. (2007). Restriction Enzymes. Retrieved from

[Link]

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