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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; GAATTC CTTAAG. This type of asymetric cut leaves a single stranded sequence hanging on either 5 or 3 end. Created on 04/02/2002 08:30:00

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 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 37C in a small volume (e.g., 20 l) 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 (), 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 (-20C 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 37C, 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) 14l distilled water 2l 10 X EcoRI Buffer 3l of pUC19 DNA (uncut) ll 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 37C for 20 minutes (in a 37C 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: 14l distilled water 2l 10 X EcoRI buffer 3l pRAS2 DNA (uncut) ll 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 37C 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: 30l distilled water 5l 10 X EcoRI Buffer l0l Wheat germ DNA (prepared in earlier exercise) 5l 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 37C waterbath for several hours. (note- the instructor may have to complete this and prepare a sample for electrophoresis to be done the next day) Created on 04/02/2002 08:30:00

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: 14l distilled water 2l 10 X EcoRI Buffer 3l Lambda DNA (uncut) 1l 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 37C (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 2l of loading dye- Label this tube pUC19/EcoRI. Place this tube on ice. 3. When the incubation of the pRAS2 digest ends, add 2l of loading dye directly to this digestflick 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 2l 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 2l 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 20l of each sample (the one kilobase ladder and 4 digests) into the wells in the order indicated.

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Lane 1 - one kb ladder Lane 2 - leave empty or use for practice loading Lane 3 - genomic Wheat Germ digest 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 3l (1l/digest) 2. Restriction Enzyme EcoRI for genomic digest 5l (optional) 3. 10 X buffer for plasmid digests using EcoRI, 6l 4. 10 X buffer for genomic digest using EcoRI, 5l (optional) 5. pUC19 DNA uncut, 3l 6. pRAS2 DNA uncut, 3l 7. lambda DNA uncut, 3l 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, 20l 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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