______—Module 5 – Restriction Enzyme Analysis

PURPOSE:

MATERIALS:

PROCEDURE:

1.  Prep:

2.  Make a reaction cocktail in a 1.5ml microtest tube and mix.

150µl qualified water (A)

25µl restriction reaction buffer (O)

35µl resuspended recombinant plasmid

3.  Label four (4) 1.5ml microtest tubes 3-6. (Tubes 1 and 2 are standard DNA fragments and supercoiled vector, respectively.)

4.  Transfer 40µl of the cocktail to each tube.

5.  Add 10µl of qualified water (A) to tube 3.

6.  Add 5µl of qualified water (A) to tubes 4 and 5.

7.  Add 5 µl (10-15 units) of diluted EcoRI endonuclease (S) to tube 4. Tap or briefly vortex to mix.

8.  Add 5 µl (10-15 units) of diluted Pvu II endonuclease (T) to tube 5. Cap. Mix.

9.  Add 5 µl of diluted Pvu II endonuclease (T) to tube 6. Then with a fresh pipet tip, add 5 µl (10-15 units) of diluted Cla I endonuclease (U) to tube 6. Cap. Mix.

RE DIGESTS-Reaction Grid

Module 5 Reactions
Tube / Reaction
cocktail / Water / Eco RI / Pvu II / Cla I / Final Vol
1--1 kb Ladder, 5µl / 20 / 25µl
2-- Supercoiled plasmid / 0 / 25µl
3- (-) control / 40 µl / 10µl / 50µl
4- Eco RI / 40 µl / 50µ
5- Pvu II / 40 µl / 50µ
6- Pvu II/ Cla I / 40 µl / 50µ

10.  Incubate tubes 3-6 at 37ºC for 1-2 hr hour.

11.  After the incubation, add 10x gel loading solution to all tubes. Also add 2µl of 100µg/ml Ethidium Bromide. Mix.

12.  Prepare agarose gel and apply samples to gel analysis.


Separation of Restriction Enzyme Reactions by Electrophoresis

Prepare 1% agarose gels according to previous instructions.

Have a water bath or beaker of water warmed to 65ºC for heating the tubes containing DNA fragments before gel loading. At 65ºC, nonspecific aggregation due to sticky ends generated by restriction enzyme digestions will melt. This will result in sharp individual DNA bands upon separation by agarose gel electrophoresis.

Run the gel @100V until the Bromphenol Blue is ~1cm from the bottom of the gel.

Size Determination of DNA Restriction Fragments

This is the first step for mapping DNA restriction sites, which is to determine the size of the “unknown” DNA fragments generated after electrophoresis. The assignment of sizes for DNA fragments separated by agarose gel electrophoresis can have ± 10% margin of error. The sizes of the “unknowns” will be extrapolated by their migration distances relative to the Standard DNA Fragments (Sample A), for which the size of each fragment is known.

Measure and record the distance traveled in the agarose gel by each 1 kb Ladder DNA fragment (250 bp-10kbp).

In each case, measure from the lower edge of the sample well to the lower end of each band. Record the distance traveled in centimeters (to the nearest millimeter).

Label the semi-log graph paper:

Label the non-logarithmic horizontal x-axis “Migration Distance” in centimeters at equal intervals.

Label the logarithmic vertical y-axis “ Log base pairs”. Choose your scales so that the data points are well spread out. Assume the first cycle on the y-axis represents 100-1,000 base pairs and the second cycle represents 1,000-10,000 base pairs.

For each Standard DNA fragment, plot the measured migration distance on the x-axis versus its size in base pairs, on the y-axis.

Draw the best average straight line through all the points.The line should have approximately equal numbers of points scattered on each side of the line.

Measure the migration distance of each of the 3, 4, 5 and 6 fragments from samples.

Based on your measurements and the “best fit” line, determine the size of your fragmants and the orientation of the insert.


Project I—Module V ______

Results:

Discussion: