MOLECULAR BIOLOGY AND GENETIC ENGINEERING

MSBO209

Experiment 8

Aim of the Experiment

To determine the restriction endonuclease sites of two restriction enzymes on a circular plasmid and construct a restriction map of the plasmid.

Introduction

Restriction mapping is the process through which one can obtain the complete structural information of a plasmid by using restriction endonucleases. It involves the digestion of DNA with a series of restriction enzymes and then performing agarose gel electrophoresis method to separate the digested DNA fragments and measure the molecular size of the same.

Principle

Negatively charged DNA molecules are separated on agarose gel matrix according to their molecular weight upon electrophoresis. The position of DNA in the agarose gel is visualized by staining the gel with low concentration of a fluorescent intercalating dye like Ethidium bromide. Smaller molecules move faster and migrate farther than larger ones because the migration rates of DNA molecules are inversely proportional to the logarithms of the molecular weights. This method is frequently performed to determine the size of an unknown DNA fragment by comparing it with DNA ladders of known size. A standard curve can be obtained by plotting the molecular size of the fragments of the marker against the reciprocal of their respective mobility. The relative mobility (Rf) of the DNA ladder depends upon the log of its relative molecular weight. The Rf value can be determined after dividing the distance traveled by the DNA by distance traveled by tracking dye. The molecular size of the test sample can be obtained from the standard curve. The mobility rate of DNA molecules vary from one experiment to another. So, the control DNA ladder should always be loaded onto the same gel.

To make a restriction map of a circular plasmid, (with three restriction sites for restriction enzyme X and two restriction sites for restriction enzyme Y) it has to be digested with two restriction enzymes separately as well as together and after that agarose gel electrophoresis has to be performed to determine the size of the digested fragments as shown in figure 1.

From the gel picture the following can be inferred:

After restriction digestion with X, the sizes of the fragments are: 6 kb, 3 kb and 1 kb.
After restriction digestion with Y, the sizes of the fragments are: 5.5 kb and 4.5 kb.
After restriction digestion with both X and Y, the sizes of the fragments are: 4 kb, 2.5 kb, 2 kb, 1 kb and0.5 kb.

With the above data, the restriction map of the plasmid can be done in the following way:

a. First draw a circle

b. Add up the fragments from each of the restriction profiles. Each should add upto the same number.

X = 6 kb+ 3 kb + 1 kb = 10 kb
Y = 5.5 kb + 4.5 kb = 10 kb
X & Y = 4 kb + 2.5 kb + 2 kb + 1 kb + 0.5 kb =10 kb

c. Put in the first site at 12 O’ clock position.

d. Put in the remaining restriction enzyme (X) sites according to their sizes.

e. From the gel digest, there are three fragments which means there are 3 sites as marked below:

f. Put in sites for the second enzyme Y and map these sites relative to the X sites.

The second enzyme generates two fragments 5.5 kb and 4.5 kb, so there must be two sites.
The combination of the two enzymes X and Y generates fragments of 4 kb, 2.5 kb, 2 kb, 1 kb and 0.5 kb.
The final map that we obtain as follows:

Fig 1: The pattern of the digested DNA after gel electrophoresis.

Fig 2: X

Fig 3: X and Y

Materials Required

Glasswares: Measuring cylinder, Beaker

Reagents: Ethidium bromide (10 mg/ml), Distilled water

Other requirements: Electrophoresis apparatus, UV Transilluminator, Heating block or Water Bath, Vortex Mixer, Micropipettes, Tips, Adhesive tape, Crushed ice, Microwave/ Hotplate/ Burner.

Procedure:

1. Before starting the experiment, crush ice and place the vials containing plasmid DNA, Restriction Enzymes and Assay Buffers onto it.

2. In this experiment plasmid DNA is digested with two restriction enzymes EcoRI and HindIII separately as well as together.

3. Set up the reaction mixture as follows:

Reaction 1 (EcoRI digestion) :

1. Plasmid DNA – 5.0 μl

2. 10X Assay Buffer – 5.0 μl

3. Molecular Biology Grade Water – 39.0 μl

4. EcoRI –1.0 μl

Total 50μl

Reaction 2 (HindIII digestion)

1. Plasmid DNA – 5.0 μl

2. 10X Assay Buffer – 5.0 μl

3. Molecular Biology Grade Water – 39.0 μl

4. HindIII – 1.0 μl

Total 50μl

Reaction 3 (EcoRI & HindIII digestion)

1. Plasmid DNA – 10.0 μl

2. 10X Assay Buffer – 5.0 μl

3. Molecular Biology Grade Water – 33.0 μl

4. HindIII –1.0 μl

5. EcoRI – 1.0 μl

Total 50μl

4. After preparing the three reaction tubes, mix the components by gentle pipetting and tapping.

5. Incubate the tubes at 37oC for 2 hours.

6. After incubation, immediately add 10 μl of 6X gel loading buffer to each tube.

7. Run the samples on agarose gel as given below.

Agarose Gel Electrophoresis:

1. Prepare gel tray by sealing the ends with adhesive tape. Place comb in gel tray about 1 inch from one end of the tray and position the comb vertically such that the teeth are about 1-2 mm above the surface of the tray.

2. To prepare 50 ml of 1.0 % agarose solution, measure 0.5 g agarose into a glass beaker or flask and add 50ml of 1X TAE buffer. Heat the mixture on a microwave or hot plate. Swirl the glass beaker/flask occasionally, until agarose is dissolved completely (Ensure that the lid of the flask is loose to avoid buildup of pressure).

3. Allow the solution to cool down to about 55-60C. Add 2 μl of Ethidium bromide (10 mg/ml), mix well and pour the gel solution into the gel tray to a depth of about 5mm. Allow the gel to solidify for about 30 minutes at room temperature.

4. To start the run, carefully remove the adhesive tape from both the ends of the gel tray, place the tray in electrophoresis chamber, and fill the chamber (just until wells are submerged) with 1X TAE electrophoresis buffer and gently remove the comb.

5. Load 3 μl of the ready to use DNA ladder into well 1. Load 15 μl of each DNA samples from Reaction I and Reaction 2 onto wells 2 and 3. Load 30 μl of DNA samples from reaction 3 onto well 4.

6. Connect power cord to the electrophoretic power supply according to the convention: Red-Anode and Black- Cathode. Electrophorese at 100-120 volts and 90 mA current until dye markers have migrated an appropriate distance, depending on the size of DNA to be visualized.

7. Electrophoresis apparatus should always be covered to protect against electric shocks. Avoid use of very high voltage which can cause trailing and smearing of DNA bands in the gel, particularly with high-molecular-weight DNA.

8. Monitor the temperature of the buffer periodically during the run. If the buffer becomes heated, reduce the voltage. Melting of an agarose gel during electrophoresis is a sign that the voltage is too high, that the buffer may have been incorrectly prepared or has become exhausted during the run.

9. Switch off the power supply once the tracking dye from the wells reaches 3/4th of the gel which takes approximately 45 minutes. Observe the gel on a UV transilluminator.