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Goal: To introduce the plasmid DNA samples to TaqI, BsmI, and

FokI restriction enzymes to determine if polymorphisms are present
in the Vitamin D receptor.
Reagents:
 Plasmid DNA
 4-Core Buffer System, 10X (TaqI)
 CutSmart 10X Buffer (BsmI, FokI)
 TaqI restriction enzyme
 BsmI restriction enzyme
 FokI restriction enzyme
 600 µL microcentrifuge tubes
 Gel (1.5% agarose in TAE buffer)
 Casting tray and comb
 Electrophoresis apparatus
 Loading Buffer
 Tupperware dish
 1X Sybrgreen

Before Starting
1. Fill an ice bucket for plasmid DNA samples
Important: Remember to keep the restriction enzymes and DNA samples on ice
at all times!

Methods: Restriction Enzyme Digestion

TaqI Enzyme Digestion
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1. Add the following reagents to a 600 µL microcentrifuge tube:

Reagent [Initial] Volume Added [Final] u/µL

Plasmid DNA 46 µL
RE Buffer 10X 5.2 µL 1X
TaqI restriction 1 µL 10
enzyme (ADD
LAST)
Total Volume 52.2 µL
This solution consists of 1 part 10X buffer, 9 parts enzyme/DNA, so the final concentration of the buffer is
1X.
2. Incubate for __hours or O/N at 37°C

BsmI Enzyme Digestion

1. Add the following reagents to a 600 µL microcentrifuge tube:

Reagent [Initial] Volume Added [Final] u/µL
Plasmid DNA 46 µL
CutSmart Buffer 10X 5.2 µL 1X
BsmI restriction 1 µL 10,000
enzyme (ADD
LAST)
Total Volume 52.2 µL

2. Incubate for __hours or O/N at 37°C

FokI Enzyme Digestion

1. Add the following reagents to a 600 µL centrifuge tube:

Reagent [Initial] Volume Added [Final] u/µL

Plasmid DNA 46 µL
CutSmart Buffer 10X 5.2 µL 1X
FokI restriction 1 µL 5,000
enzyme (ADD
LAST)
Total Volume 52.2 µL

2. Incubate for __hours or O/N at 37°C

Methods: Gel Analyzation of the Restriction Enzyme Digestion

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1. Gel preparation—place a comb at one end of the casting tray to form wells
for the samples in the DNA agarose gel.
2. Let the agarose sit for 10 minutes to solidify.
3. Remove the barriers from the ends of the tray and move the gel to an
electrophoresis apparatus. Remove the comb.
4. Pour TAE buffer into the apparatus such that it fills the reservoirs on the
sides of the gel and covers it (~0.5cm above the gel).
5. Preparation for electrophoresis—dilute the samples with loading buffer (see
the table below). This dilution will dilute the buffer solution from 6X to 1X.

Note: Label new tubes and add the sample and buffer combinations to their
respective tubes. Also determine the order of samples for the gel tray (numbered
left to right).
Reagent [Initial] Volume Added [Final]
Loading Buffer 6X 10.4 µL 1X
Sample 52.2 µL
Total Volume 62.6 µL

6. Heat the sample with the 1X loading buffer for 5 minutes at 60°C. Take this
time to prepare the known DNA size standards.
7. Immediately load the samples into the wells into the determined order.
8. After loading is complete, electrophoreses the DNA with a voltage of 100V.
a. Be sure to check that bubbles are rising (this indicates the current
running through the buffer)
b. Check that the dye is moving through the gel in the correct direction
c. Check the gel at least within the first 30 of electrophoresis
9. Continue electrophoresis until the dye front has reached ¾ of the length
through the gel.
10. WEAR GLOVES—transfer the gel to a Tupperware dish. Incubate in 1X
Sybrgreen in TAE buffer for 15 minutes on the rotator.
11. WEAR GLOVES—transfer the gel to a TAE buffer that doesn’t contain
Sybrgreen and incubate for 5 minutes on the rotator.
12. WEAR GLOVES—place the gel on a UV transilluminator. Replace the
protective shield, then turn the UV lights on and the lab lights off. Take a
picture of the DNA fragments with a smartphone.
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Note: if the DNA fragments are not separated enough, return the gel to the
electrophoresis apparatus and apply voltage, allowing the dye to migrate
farther through the gel.

Note: undigested plasmid DNA may run as two separate bands—circular

DNA (partially cut, the slowest) or superhelical circular DNA (uncut)

13. Record the findings, including the size of the known DNA fragments and
the distance they migrated through the gel (measure from the middle of the
well to the middle of the DNA fragment). Use this data to create a linear line
graph, which will allow for the determination of the sample fragment sizes.

Results:
Conclusion:
Future Experiments: