_______—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
1--1 kb
Ladder, 5µl
2-Supercoiled
plasmid
Reaction
cocktail
Water
Eco
RI
Pvu
II
Cla
I
Final Vol
20
25µl
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: