Laboratory Exercise-RESTRICTION ENZYMES

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					                      Laboratory Exercise-RESTRICTION ENZYMES
                           (modified by Donald Holmquist, 2001)


In this exercise you will;
1. Digest two plasmids with EcoRI
2. Digest wheat germ DNA with EcoRI (optional)
3. Digest Lambda DNA with EcoRI
4. Analyze all digests with gel electrophoresis

Introduction
Recombinant DNA technology or “gene splicing,” as it is frequently called, often involves
insertion of selected DNA sequences from a variety of sources into plasmids which are then
transformed into bacteria. The resultant recombinant bacterial strain may now express the
plasmid genes as well as whatever foreign gene(s) that was inserted.
This process would not be possible without accurate and predictable ways of cutting the DNA of
the plasmid and the foreign source. Restriction endonuclease enzymes provide this service.
Restriction endonucleases, often called restriction enzymes, cut DNA within the molecule by
hydrolyzing the phosphodiester bonds between the nucleotides. Restriction enzymes were
discovered in bacteria and there are now more than 1200 known restriction enzyme types. These
enzymes are named using a simple system. EcoRI, for example, was isolated from E. coli and
was the first enzyme isolated from a particular strain, hence the designation of I. HaeIII was
isolated from Haemophilus aegyptius. HindIII was isolated from Haemophilus influenzae, and
was the third enzyme discovered in a particular strain.

Usually, restriction enzymes only cut the DNA at or near a very specific nucleotide sequence
known as a recognition site. This “restrictive” nature of these enzymes allows molecular
biologists to pin-point exactly where the DNA is to be cut. In many cases, the recognition site of
a restriction enzyme consists of a palindromic sequence where the order of the bases is read the
same on each side of the helix. The two sides of the helix are antiparallel and the recognition
sequence reads from 5’ to 3’ on either side of the helix. For example the recognition site for
EcoRI is as follows;

                                  GAATTC
                                  CTTAAG

Some restriction enzymes cut both sides of the helix at the same point. HaeIII is such an enzyme
and the fragments produced are “blunt” ended. Many restriction enzymes make asymetric cuts
in the helix. EcoRI cuts DNA at the recognition site in the following fashion;

                                   G↓AATTC
                                   CTTAA↑G.

This type of asymetric cut leaves a single stranded sequence hanging on either 5’ or 3’ end.
Such a trailing end is often called a “sticky” end since it can bond by hydrogen bonding to a
complementary single stranded sequence. The sticky ends make it rather easy to insert DNA into


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a plasmid. If the plasmid and DNA to be inserted are cut with the same restriction enzyme, the
sticky ends will match and will anneal using DNA ligase enzyme. The result is a recombinant
plasmid with a foreign fragment incorporated. Cleavage of DNA by a restriction enzyme is
called digestion and is usually carried out at 37˚C in a small volume (e.g., 20 μl) of buffer and
salt. Although restriction enzymes are active over a fairly broad range of conditions, optimal
conditions have been determined for each restriction enzyme using a buffer which typically
contains 10-100mM Tris-HCl pH 8.0, a salt (usually 0-150 mM NaCl), and 10 mM of
Magnesium Chloride. Restriction enzyme digestion involves adding enough restriction enzyme
to the substrate DNA in the appropriate buffer and letting the digest incubate at the appropriate
temperature for at least one hour. Restriction enzymes are measured functionally in units (μ),
which are defined as the amount of restriction enzyme that will completely digest a defined
substrate DNA in a defined time at a specific temperature. When stored at the recommended
conditions (-20˚C in storage buffer containing 50% glycerol), restriction enzymes can remain
active for many months. However, once they are diluted into digestion buffer and incubated at
37˚C, they lose activity within hours.




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  Procedure:

  Part 1= EcoRI digests

  A. Restriction Enzyme Digestion of pUC19 DNA
  (note- all components involved in digestion should be kept on ice)
  1. Prepare an EcoRI digest of pUC19 DNA by adding the required components to a clean
     microtube in the following order: (ORDER IS CRITICAL)
     14μl distilled water
     2μl 10 X EcoRI Buffer
     3μl of pUC19 DNA (uncut)
     lμl of Restriction enzyme EcoRI
  Place your initials on this tube and label it pUC19/EcoRI
  2. Flick the tube to mix well and spin for 5 sec in the microfuge to bring all the components to
     the bottom.
  3. Incubate this digest mixture at 37C for 20 minutes (in a 37C waterbath).

  B. Restriction Enzyme Digestion of pRAS2 DNA
  (note- all components must be kept on ice)
  1. Prepare an EcoRI digest of pRAS2 DNA by adding the components to a clean microfuge tube
     in the following order:
     14μl distilled water
     2μl 10 X EcoRI buffer
     3μl pRAS2 DNA (uncut)
     lμl Restriction enzyme EcoRI
  Place your initials on this tube and label it pRAS2/EcoRI
  2. Flick the tube and spin for 5 seconds in the microfuge to bring all the components to the
     bottom.
  3. Incubate at 37C in an incubator or waterbath for 20 minutes.

  C. Restriction Enzyme Digestion of Genomic Wheat Germ DNA using EcoRI
    (optional)
   (note- keep all components on ice)
   1. Prepare an EcoRI digest of genomic Wheat Germ DNA by adding the components to a clean
      microtube in the following order:
      30μl distilled water
      5μl 10 X EcoRI Buffer
      l0μl Wheat germ DNA (prepared in earlier exercise)
      5μl Restriction Enzyme EcoRI
   Place your initials on this tube and label it genomic/EcoRI
2. Flick the tube and spin it for 5 seconds in the microfuge to bring all components to the bottom.
3. Incubate this digest mixture in a 37C waterbath for several hours. (note- the instructor may have
   to complete this and prepare a sample for electrophoresis to be done the next day)




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D. Restriction Enzyme Digestion of Lambda phage DNA using EcoRI (note - all
  components must be kept on ice)
1. Prepare an EcoRI digest of Lambda DNA by adding the components to a clean microtube in the
   following order:
      14μl distilled water
      2μl 10 X EcoRI Buffer
      3μl Lambda DNA (uncut)
      1μl Restriction enzyme EcoRI
   Place your initials on this tube and label it Lambda/EcoRI
   2. Flick the tube and spin for 5 second in the microfuge to bring all the components to the
      bottom.
   3. Incubate this digest mixture at 37C (waterbath) for 20 minutes.

  Part II -Preparation of samples for electrophoresis

  1. When the incubation of the pUC19 digest ends, transfer this tube to ice for 5 minutes.
  2. Add 2μl of loading dye- Label this tube pUC19/EcoRI. Place this tube on ice.
  3. When the incubation of the pRAS2 digest ends, add 2μl of loading dye directly to this digest-
     flick the tube to mix and microfuge for a few seconds. Transfer this tube to ice.
  4. When the incubation of the genomic Wheat Germ DNA digest ends, transfer this tube to ice
     for 5 minutes.
  5. Add 2μl of loading dye. Flick this tube and briefly spin and label it Genomic/EcoRI. Place this
     tube on ice.
  6. When the incubation of the Lambda DNA digest ends, transfer this tube to ice for 5 minutes.
  7. Add 2μl of loading dye. Label this tube Lambda/EcoRI and place on ice.

  END OF FIRST DAY (Double Period)

  Note: At this point each group should have 4 tubes (4 digests) which contain loading dye. These
  tubes can be stored on ice overnight.

  Review www-hhmi.Princeton.edu/99grp7/ for photographic details of gel electrophoresis
  procedure.

  Part III - Electrophoresis of EcoRI digests (day two)

  1. Prepare a 1% Agarose Gel (eight well comb) and allow it to stand for 20 minutes
  2. After removing the dams, add the chamber buffer. Then, after a few minutes to allow the gel
     to soften, remove the comb by pulling straight up. (Note- The instructor can save time by
     doing steps 1 and 2 prior to class),
  3. Load 20μl of each sample (the one kilobase ladder and 4 digests) into the wells in the order
     indicated.

      Lane 1 - one kb ladder
      Lane 2 - leave empty or use for practice loading
      Lane 3 - genomic Wheat Germ digest


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   Lane 4 - pUC19 digest
   Lane 5 - pRAS2 digest
   Lane 6 - lambda digest
   Lane 7- lambda digest (HindIII) if available

4. After the samples have been loaded, close the lid, connect the power supply, and switch on.
   Set the voltage at 110 volts.
5. Electrophorese for about 30 minutes until the dye has reached midway between the second and
   third red stripes. Switch off the power supply and open the lid.
6. Wearing gloves, remove the gel and stain with methylene blue in a tray for 30 minutes and
   move the gel to a tray of distilled water for 15 minutes to destain.
7. After staining, place the gel in a tray (half of an empty tip box ) using a spatula and bring to
   the light box.
8. The instructor will photograph your gel. (You may destain overnight if needed.)
9. After photographing, discard the gel and gloves in the waste container for proper disposal.

Discussion Questions
1. How many places does EcoRI cut pUC19? How many fragments are produced?
2. Approximate the size of the pUC19 fragment(s) using the kilobase ladder. Your instructor will
   show you a graphing method using semi-log paper.
3. How many places does EcoRI cut pRAS2? How many fragments are produced? Approximate
   the sizes of these fragments.
4. pRAS2 is a modification of pUC19 in which a segment of yeast DNA is added. Do your
   results support this? Explain?
5. The digest of Lambda DNA by EcoRI produces 6 fragments of the following sizes; 21226 bp,
   7421 bp, 5804 bp, 5643 bp. 4878 bp, 3530 bp. The 5804 and 5643 fragments may appear as a
   single heavy band. Why? Using the graphing technique and the one kilobase ladder bands and
   their migration distances, approximate the sizes of the lambda fragments and compare these to
   the actual sizes listed above. Determine a percent error for each fragment measurement.
6. How many fragments are produced by digestion of your genomic Wheat Germ DNA
   byEcoRI? Is this what you expected? Explain.




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Teacher Preparation Guide

1. This exercise requires one double period and a single period the next day (or within a few
   days). Each student group should contain four students. One student member of each group
   should work on one digest and follow all the procedures for that digest up to and including
   preparation for electrophoresis. (There are four digests- pUC19, pRAS2, lambda phage, and
   genomic (if you isolated this in a previous lab). All digests should be prepared, incubated,
   prepared for electrophoresis and stored the first day.
2. The gels should be made the second day and may be poured ahead of time and stored under
   chamber buffer.
3. The gels may not be finished running during the class period of the second day, so the teacher
   may have to stain the gels and photograph them. The gels should be stained and
   photographed right after being run since the bands become diffuse with time.
4. As always, make certain that the students are familiar with all transfer techniques,
   electrophoresis procedures and care and cleaning of equipment. Stress safety when dealing
   with high voltage.

Materials List for Restriction Enzyme Exercise (for each group of four students)

   1. Restriction enzyme EcoRI for plasmid digests 3μl (1μl/digest)
   2. Restriction Enzyme EcoRI for genomic digest 5μl (optional)
   3. 10 X buffer for plasmid digests using EcoRI, 6μl
   4. 10 X buffer for genomic digest using EcoRI, 5μl (optional)
   5. pUC19 DNA uncut, 3μl
   6. pRAS2 DNA uncut, 3μl
   7. lambda DNA uncut, 3μl
   8. microtubes, 0.65 ml clear (as needed)
   9. microfuge, 10k with 2 rotors
   10. ice
   11. Float for water bath
   12. Water Bath, 37 C
   13. Loading dye - as needed
   14. p20 and tips
   15. marking pen
   16. one kilobase ladder, 20μl
   17. TE Buffer, 0.1 ml
   18. Electrophoresis chamber, gel casting tray, combs, dams, power supply and cords
   19. l% Agarose – 25 ml
   20. gloves
   21. methylene blue solution
   22. light table and photodyne camera
   23. staining and destaining trays




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