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21-1185, 21-1187

Restriction Enzymeand DNA Kit

Teacher’s Manual

Contents

Background 3

Overview 3

Restriction Enzymes 4Lambda DNA 5Gel Electrophoresis 6

Materials 7

Scheduling 9

Pre-Lab Preparation 10

Student Lab Briefing 13

Fine Points of Lab Procedure 14

Lab Procedure 17

Results and Discussion 19

Field Guide to Electrophoresis Effects 22

Student Instructions 24

©1998 Carolina Biological Supply Company Printed in USA

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In this experiment, DNA from the bacteriophage lambda (48,502 base pairs inlength) is cut with restriction enzymes and the resulting restriction fragmentsare separated using gel electrophoresis. Three samples of lambda DNA areincubated at 37° C, each with one of three restriction endonucleases: BamHI,EcoRI, and HindIII. A fourth sample, the negative control, is incubated withoutan endonuclease. The DNA samples are then loaded into wells of an agarosegel and electrophoresed. An electric field applied across the gel causes theDNA fragments in the samples to move from their origins (sample wells)through the gel matrix toward the positive electrode. Smaller DNA fragmentsmigrate faster than larger ones, so restriction fragments of differing sizesseparate into distinct bands during electrophoresis. The characteristic numberand pattern of bands produced by each restriction enzyme are, in effect, a“DNA fingerprint.” The restriction patterns are made visible by staining with acompound that binds to DNA.

BackgroundNote: The following background information is not included in the StudentWorksheets; however, an abbreviated version of this background material isincluded in the Dry Lab Student Activities.

OverviewBiotechnology is actually a collection of many different technologies, all ofwhich use cells and biological molecules to solve problems and make usefulproducts. One of these types of technologies is recombinant DNA technolo-gy. Recombinant DNA is made by joining or recombining genetic materialfrom two different sources. This happens frequently in nature, in order toincrease genetic variation. However, recombinant DNA can also be madeartificially, in the laboratory. This was first made possible by the discoveryof restriction enzymes. Restriction enzymes made it possible for scientiststo cut DNA into small, defined pieces that were easy to work with. LambdaDNA was frequently used in early studies of DNA since it has a relativelysmall genome. Gel electrophoresis is the technique chosen here to visualizethe DNA.

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Restriction Enzymes

Restriction enzymes are proteins produced by bacteria to prevent or restrictinvasion by foreign DNA. They act as DNA scissors, cutting the foreign DNAinto pieces so that it cannot function. Restriction enzymes recognize and cutat specific places along the DNA molecule known as restriction sites. Eachdifferent restriction enzyme (and there are hundreds, made by many differ-ent bacteria) has its own type of site. In general, a restriction site is a 4- or 6-base-pair (bp) sequence that is a palindrome. A DNA palindrome is asequence in which the “top” strand read from 5’ to 3’ is the same as the“bottom” strand read from 5’ to 3’. For example,

5’ GAATTC 3’

3’ CTTAAG 5’

is a DNA palindrome. To verify this, read the sequences of the top strand andthe bottom strand from the 5’ ends to the 3’ ends. This sequence is also arestriction site for the restriction enzyme called EcoRI. The name EcoRI comesfrom the bacterium in which it was first discovered—Escherichia coli RY 13(EcoR) and I, because it was the first restriction enzyme found in this organ-ism.

EcoRI makes one cut between the G and the A in each of the DNA strands(see below). After the cuts are made, the DNA is held together only by thehydrogen bonds between the four bases in the middle. Hydrogen bonds areweak, and the DNA comes apart.

Cut DNA: 5’ G AATTC 3’

3’ CTTAA G 5’

As you can see, when EcoRI cuts a DNA molecule it leaves single-stranded“tails” on the new ends. This type of end is called a “sticky end” because it iseasy to rejoin it to complementary sticky ends. Not all restriction enzymesmake sticky ends; some cut straight across the DNA molecule, producing ablunt end. When scientists study a DNA molecule, one of the first things theydo is figure out what restriction sites are present, and where the restrictionsites are in the DNA molecule. A restriction enzyme digest of a particular DNAmolecule produces a distinctive pattern of DNA fragments, specific to that DNAmolecule, which can then be seen with gel electrophoresis. The DNA moleculeyou will be studying in this experiment is lambda bacteriophage DNA, and therestriction enzymes you will be using are BamHI, EcoRI, and HindIII.

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The restriction sites for each of these enzymes is shown below:

BamHI 5’ G GATCC 3’3’ CCTAG G 5’

EcoRI 5’ G AATTC 3’3’ CTTAA G 5’

HindIII 5’ A AGCTT 3’3’ TTCGA A 5’

Lambda DNA

Lambda is a bacteriophage, a virus that only infects bacteria. Lambda infectsE. coli bacteria. Its DNA is a double-stranded molecule of 48,502 bp. LambdaDNA can exist as either a linear or circular molecule. Circularization of lambdaDNA depends on 12-nucleotide single-strand complementary sequences ateach end of linear lambda DNA. These single-stranded DNA regions arereferred to as the cos (cohesive) site, and they are essential for packaging ofDNA inside the phage. When the single-stranded ends form base pairs, thelambda DNA circularizes. This can create some confusion when interpretingrestriction digests of lambda DNA. To remedy this situation, the DNA can beheated to 65° C for 3 min immediately prior to electrophoresis. This will breakthe hydrogen bonds holding the cos site together.

The table below shows a partial list of recognition sites for restriction enzymesthat cleave lambda DNA and the lengths of fragments that are generated fromlinear lambda DNA. The first base at the 5’ end of the linear DNA is designatednucleotide number one.

Restriction Number Positions LengthsEnzyme of Sites of Sites of FragmentsBamHI 5 5505, 22346, 5505, 5626, 6527,

27972, 34499, 6770, 7233, 1684141732

EcoRI 5 21226, 26104, 31747, 3530, 4878, 5643,39168, 44972 5804, 7421, 21226

HindIII 7 23130, 25157, 27479, 125, 564, 2027,36895, 37459, 37584, 2322, 4361, 6557,44141 9416, 23130

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Gel Electrophoresis

Electrophoresis means literally to carry with electricity. It is a widely-usedbiotechnology technique used to separate charged molecules such as DNA,RNA, and proteins.

Electrophoresis is frequently performed using an agarose gel. Agarose is apolysaccharide like agar or pectin that dissolves in boiling water and thengels as it cools. In electrophoresis, the sample is applied to a slab of gelledagarose and then an electric current is applied across the gel. Agarose gelsmust be prepared and run in a buffer.

The buffer is necessary because ions would otherwise cause the anode tobecome alkaline and the cathode to become acidic. The buffer you have istris-borate-EDTA (TBE). Remember that a buffer is a mixture of a weak acid orbase and its salt. Tris is a weak base, and the Tris salt is made by adding boricacid. The metal chelator EDTA (ethylenediaminetetraacetic acid) is also added.By chelating (specifically binding to) calcium ions, EDTA inhibits RNases andDNases, which could degrade RNA and DNA. It is important to have theproper concentration of buffer. In the absence of ions (e.g., if the gel weremistakenly run in water) electrical conductance is minimal. On the other hand,if the buffer is too concentrated (e.g., if 10× buffer were used by mistake)electrical conductance is too efficient and heat is generated. Too much heatwill melt the gel and denature the DNA.

When an electric field is present, a negatively charged sample (such asDNA) migrates through the gel toward the positive electrode. The rate ofmigration of a DNA molecule depends on the size and also on whether themolecule is circular or linear. The voltage applied to the agarose gel alsoinfluences how quickly a sample moves through the gel. The higher the volt-age, the more quickly the sample moves. However, there is a trade-offbecause at higher voltages samples do not separate with as much resolution.Also, if the voltage is too high, the gel will become hot and melt.

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MaterialsWarning: Individuals should use this kit only in accordance with prudent lab-oratory safety precautions and under the supervision of a person familiarwith such precautions. Use of this kit by unsupervised or improperly super-vised individuals could result in serious injury.

The materials in the DNA Restriction Analysis Kit are sufficient for six com-plete setups of the experiment. The materials are supplied for use with theexercise in this kit only. Carolina Biological Supply Company disclaims allresponsibility for any other uses of these materials. See specific warnings intext.

This kit contains proprietary dehydrated formulations of DNA and restrictionenzymes developed by Life Technologies, Inc. All materials in the kit can bestored at room temperature for up to 2 years. The DNA and restrictionenzymes are shipped in a resealable foil pouch with a dessicant. Storeunused tubes of DNA and enzymes in the pouch with the dessicant. Oncethe DNA is reconstituted, it should be stored in the refrigerator.

To reconstitute the DNA, add distilled or deionized water as described in theenclosed directions. The restriction enzymes are dried with restriction buffer insingle-use reaction tubes. Restriction digests are set up by adding rehydratedDNA to an enzyme tube and resuspending the dried enzyme and buffer.

Materials included in this kit:

Vials Lambda DNA* (clear)

6 Vials BamHI (blue)

6 Vials EcoRI (pink)

6 Vials HindIII (green)

6 Vials Loading Dye (100 µL)

TBE Buffer Concentrate (150 mL)

6 Control Reaction Tubes (0.2mL)

(yellow)

Dry Labs:

DNA Scissors

DNA Goes to the Races

*(2 vials if using ethidium bromide and 3 vials if using Carolina BLU™)

**The ethidium bromide kit includes ethidium bromide stain at 1 µg/mL and 250 mL0.05 M KMnO4 for decontamination. The Carolina BLU™ kit contains 7.5 mL of gel andbuffer stain and 250 mL of final stain.

2 Pairs of Disposable Gloves

6 Staining Trays

Stain** (250 mL)

15 1.5-mL Tubes

Instructor’s Manual

Student Guides

Agarose (3.2 g, for 400 mL)

Floating Tube Rack

Materials needed, but not provided:

6 Micropipettors (0–10 µL or 0–20 µL)

50 Micropipet Tips

6 Gel Electrophoresis Chambers and Power Supplies

Flask or Beaker for Agarose (1 L)

Carboy or Container for Electrophoresis Buffer (3 L or Larger)

Distilled Water

6 Permanent Laboratory Markers (felt tip or wax)

Toothpicks

Boiling Water Bath or Microwave Oven

Water Bath at 60° C

Water Bath at 37° C

Funnel

Note: Students need semilog graph paper and metric rulers for Question 4of the Results and Discussion section.

Carolina BLU™ was developed as a significant advance over the methyleneblue staining protocol and is provided at 2 concentrations. With the CarolinaBLU™ method, small amounts of concentrated Carolina BLU™ gel and bufferstain are added to the agarose and electrophoresis buffer. During elec-trophoresis, Carolina BLU™ faintly stains the DNA, allowing for its immediatevisualization. This allows students to view their results without the lengthystaining and destaining steps required for methylene blue. Following elec-trophoresis, the DNA bands can be stained more intensely by soaking thegel in a dilute solution of Carolina BLU™ for 15–20 min. At this stage theDNA bands are stained more intensely. Background stain in the gel can beremoved by several washes in deionized water over a 30–40 min period.This additional step intensifies the staining of all the fragments, making thesmaller fragments more visible than is usually possible with methylene bluestain. If you do not wish to add stain to the agarose and buffer, the finalstain will still work as does methylene blue. The advantages of the CarolinaBLU™ gel system over methylene blue staining are:

1. Immediate visualization of DNA.

2. Greatly shortened staining and destaining times.

3. Superior results. When used as described, Carolina BLU™ stains DNAmore intensely, allowing for easier visualization of all DNA bands andvisualization of smaller bands which are not usually seen with methyl-ene blue staining.

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The kit can be stored at room temperature for up to 2 years. Volumes of agarosesolution and TBE buffer provided are sufficient for most gel systems using 10-cm gels, such as Carolina Biological Supply Co.’s #21-3668.

A white-light box is desirable for viewing blue-stained gels, although gels canalso be viewed on an overhead projector. A Polaroid® “gun” camera or othercamera is desirable for recording results. A mid- or long-wavelength UV lightsource, with protective screen or glasses, is needed to view ethidium bromide-stained gels. A 10% solution of household bleach is needed forcleanup of lab surfaces, and 0.25 N HCl and 0.25 N NaOH are required fordecontaminating ethidium bromide-staining solution and gels.

SchedulingDNA restriction analysis requires several different activities. Plan your timeas follows. If a double period is available, Lab Day 1 and Lab Day 2 can becompleted at once.

TimeDay Needed ActivitySeveral days 30 min Pre-lab: Mix TBE bufferbefore lab Reconstitute DNA & aliquot samples

Lab Day 1 30 min Pre-lab: Add stain to TBE bufferPrepare agarose solutionPool small volumes prior tosetting up work stations

15 min Lab: Practice pipetting15 min Set up restriction digest10 min Cast agarose gel20+ min Post-lab: Incubate reactions

Lab Day 2 15 min Lab: Practice loading gel15 min Load gel40+ min Post-lab: Electrophorese40 min Stain gels

Lab Day 3 40 min Results and Discussion

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Pre-Lab PreparationMix TBE Buffer

Because tris-borate-EDTA (TBE) buffer solution is stable, it can be madeahead of time and stored in a carboy or other container until ready to use.Pour contents of 20× TBE concentrate into a 3-liter flask or carboy. Add2,850 mL of distilled or deionized water for a final volume of 3 L. If there isany precipitate in the bottle containing the 20× TBE Buffer, rinse with a por-tion of the 2,850 mL of distilled water. Stir for 1–2 min.

Reconstitute DNA

If Staining with Carolina BLU™

1. Add 160 µL of distilled or deionized water to each of the 4 tubes of DNA provided. Treat the tabs on the cap gently to avoid breaking them.

2. Allow to sit for 5 min.

3. Hold the closed tube firmly at the top and flick the side of the tuberepeatedly to mix the contents. Do this for 1 full min. Repeat processfor each of the tubes of DNA.

4. Allow the tube to stand for an additional 5 min. The DNA solutionshould look slightly opaque. Note: It is very important that the DNA betotally dissolved.

5. Aliquot 90 µL of DNA into 6 1.5-mL tubes to be used by each lab group.

If Staining with Ethidium Bromide

1. Add 280 µL of distilled or deionized water to each of the 2 tubes of DNA provided. Treat the tabs on the cap gently to avoid breaking them.

2. Allow to sit for 5 min.

3. Hold the closed tube firmly at the top and flick the side of the tuberepeatedly to mix the contents. Do this for 1 full min. Repeat processfor each of the tubes of DNA.

4. Allow the tube to stand for an additional 5 min. The DNA solutionshould look slightly opaque. Note: It is very important that the DNA betotally dissolved.

5. Aliquot 90 µL of DNA into 6 1.5-mL tubes to be used by each lab group.

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Prepare Agarose Solution

If Staining with Carolina BLU™ or Ethidium Bromide

Before class on Lab Day 1, prepare 0.8% agarose solution. Add 3.2 g (entirebottle) of agarose to 400 mL 1× TBE electrophoresis buffer in a clean 1-L flaskor beaker. Cover with aluminum foil and heat in a boiling water bath (doubleboiler) for 10–20 min. Solution will become clear as agarose dissolves. Swirland observe bottom to insure that no undissolved agarose remains.Alternatively, heat solution at high setting of microwave oven for 7–10 minwithout aluminum foil. Cool solution to approximately 60° C before use. Coverwith aluminum foil and keep warm in 60° C water bath until ready to use.

If Staining with Carolina BLU™

If using Carolina BLU™ stain, the following protocol is used for the addition ofstain to the agarose and buffer.

Addition of Carolina BLU ™ to Agarose

The concentration of stain added to the agarose and buffer is dependent onthe voltage used for electrophoresis. If electrophoresing at less than 50 volts,a slightly lower concentration is utilized than if running at voltages greaterthan 50. The stain may be added to the entire volume of agarose and distrib-uted, or the agarose may be distributed to each lab station and the stainadded by the students at the rates listed below:

Voltage Agarose Volume Stain Volume

< 50 V 30 mL 40 µL (1 drop)60 mL 80 µL (2 drops)400 mL 533 µL (13 drops)

> 50 V 50 mL 80 µL (2 drops)400 mL 640 µL (16 drops)

After addition of the stain to the agarose, swirl to mix and immediately pourthe gel. Gels may be prepared 1 day ahead of the lab day if necessary. Gelsstored longer tend to fade and lose their ability to stain bands during elec-trophoresis. Store covered with a small amount of buffer, or store covered inthe gel box. Do not try using more stain than recommended in your gel. Thisleads to precipitation of the DNA in the wells and can create aggregatedDNA bands in the agarose gel.

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Addition of Carolina BLU ™ to BufferUse the chart below for addition of the stain to 1× TBE electrophoresisbuffer:

Voltage Buffer Volume Stain Volume

< 50 V 500 mL 500 µL (12 drops)2.6 L 2.6 µL (65 drops)

> 50 V 500 mL 960 µL (24 drops)2.6 L 5000 µL (125 drops)

The dropper bottle provided delivers 40 µL/drop. If a calibrated pipet is avail-able, the dropper tip can be removed for quicker addition of larger volumesof stain. The volumes of buffer and agarose required for some gel boxoptions are listed below:

Volume Buffer Volume Agarose Type Gel Box Required Required

Mini Gel System Box 200 mL 30 mL

Carolina Gel Box, 1 tray 250 mL 50 mL

Carolina Gel Box, 2 trays 450 mL 100 mL

While Carolina BLU™ is not toxic, we recommend that the students weargloves to prevent staining the skin. If reusing the buffer is important, werecommend using Carolina BLU™ in the gel and/or as final stain only.

Set Up Student Stations

Each student station should include the following materials:

Vial Lambda DNA (90 µL) Micropipet Tips

Tube Distilled Water (1 mL) Power Supply

Vial Loading Dye (100 µL) Gel Electrophoresis Chamber

Vial EcoRI (pink tube) Rack for tubes

Vial BamHI (blue tube) Masking Tape

Vial HindIII (green tube) Permanent Marker

Empty Vial (yellow tube) Staining Tray

Micropipet 2 Student Guides

1. Groups must share the following materials: agarose solution; 1× TBEelectrophoresis buffer; water bath at 37° C, and stain.

2. Hold agarose solution at 60° C in a water bath.

3. Set up a 37° C water bath for incubating restriction reactions. A constant-temperature water bath can be made by maintaining a trickle flow of tapwater into an insulated cooler. Monitor temperature with a thermometer.

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Student Lab Briefing

Pipetting Tips

Instruct students in the proper use of a digital micropipet.

• Most digital micropipets have a two-position plunger with friction“stops.” Depressing to the first stop measures the desired volume.Depressing to the second stop introduces an additional volume of airto blow out any solution remaining in the tip. On many models, athird stop ejects the tip.

• When withdrawing or expelling fluid, always hold tube firmlybetween thumb and forefinger. Hold tube at nearly eye level so youcan observe fluid level change in pipet tip.

• Do not try to pipet with test tube in rack or try to pipet into tube heldby lab partner.

• To Withdraw Sample: Depress plunger to first stop and hold in thisposition. Dip tip into solution to be pipetted and draw fluid into tip byreleasing plunger. Slide pipet tip out along inside wall of tube to dis-lodge any excess droplets adhering to outside of tip.

• To Expel Sample: Touch pipet tip to inside wall of tube into which youwant to empty sample. This creates a capillary effect that helps drawfluid out of tip. Slowly depress plunger to first stop. Depress to sec-ond stop to blow out last bit of fluid. Hold plunger in depressed posi-tion. Slide pipet out of tube with plunger depressed to avoid suckingliquid back into tip.

• To prevent cross-contaminating reactions, always use a fresh tip foreach reagent and each reaction tube.

Practice Pipetting

Students should set up the following mock reaction to practice pipettingtechnique and check their accuracy.

1. Use micropipet to add 10 µL of colored water to an empty 1.5-mL testtube. (Water may be colored with food coloring or loading dye.)

2. Pool colored water by tapping the tube bottom on lab bench, or by giv-ing the tube a short pulse in a microcentrifuge.

3. Set micropipet at 10 µL and carefully withdraw contents of tube. Isthere any air at the very end of the pipet tip? Is there any water left inthe tube? If you answered yes to either of these questions, have youover- or under-pipetted?

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Practice Gel Loading (Optional)

You may wish to have students practice loading a gel, using the directions inProcedure C. Practice gel-loading stations are available from CarolinaBiological Supply Co. (#21-1145).

Fine Points of Lab ProcedureBe aware of the following points when performing the experiment. Whereappropriate, discuss fine points with students and have them make notes ontheir Student Worksheets.

Mixing DNA with Enzymes

The enzymes in the bottom of the colored tubes contain a dye to aid in see-ing. Mix the DNA and enzymes by pipetting up and down several times.There should be no concentration of the blue color in the bottom of the tube ifthe DNA and enzymes are mixed sufficiently.

Incubating Restriction Reactions

Twenty minutes is the minimum incubation time for the restriction reaction to goto completion. If you will be electrophoresing the following day, as recommend-ed in the scheduling section, the reactions can be incubated for 1–24 hours.After several hours, enzymes lose their activity, and the reaction simply stops.Stop incubation whenever it is convenient; reactions may be stored in freezer (-20° C) until ready to continue. Thaw reactions before adding loading dye.

Storing Cast Agarose Gels

As recommended in the scheduling section, students may cast gels a day ortwo before use. If using Carolina BLU™ stain, it is best not to make the gelsmore than one day in advance. Stain fades with time, resulting in the inabili-ty to stain DNA fragments during electrophoresis. Keep gels covered withTBE electrophoresis buffer to prevent drying.

Electrophoresing

The migration of DNA through the agarose gel is dependent upon voltage—thehigher the voltage, the faster the rate of migration. Best separation is achievedwhen the bromophenol blue band from the loading dye nears the end of thegel. Do not let the bromophenol blue band run off the end of the gel. Refer tothe chart on page 15 for approximate running times at various voltages. Timesshown (hours) are for “mini-gel” system with 84-×-96-mm gel using 1.0%agarose; times will vary according to apparatus. Variations in running times for0.8% versus 1.0% gels are minor. If using the Carolina mini-gel electrophoresissystem, (catalog #21-3650) gels are run for 18–22 hours. Loading dye diffusesfrom the agarose and will be absent. Continue staining as directed.

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Carolina BLU™ Staining

Although students may stain gels in class, it saves time to stain gels afterclass, as recommended in the scheduling section. Destaining gels overnightimproves results. Wear disposable gloves during staining and cleanup.

1. Flood gels with Carolina BLU™ Final Stain, and allow to stain for 15–20 min.

2. Following staining, use funnel to decant as much Carolina BLU™ solu-tion as possible from staining tray back into storage container. Placestained gel on light box. DNA bands should be visible. If bands are faint,additional staining may be required.

3. Rinse gel in distilled or deionized water. Chlorinated water tends tobleach bands with time. Let gel soak for several minutes in severalchanges of fresh water. DNA bands will become increasingly distinct asgel destains. For best results, continue to destain overnight in a smallvolume of water. (Gel may destain too much if left overnight in large vol-ume of water.) Cover staining tray to retard evaporation.

Ethidium Bromide Staining

Warning: Ethidium bromide, like many natural and man-made substances, isa mutagen by the Ames microsome assay and is a suspected carcinogen.Stain gels after school or at another time when students are not present.With responsible handling, the dilute staining solution (1 µg/mL) used in this kitposes minimal risk. Disable and dispose of stained gels as described in Step 9.

1. Wear rubber gloves when staining gel, viewing gels, and cleaning up.

2. Confine all staining to a sink area restricted from student use.

3. Flood gels with ethidium bromide solution and allow to stain for 10 to15 min. (Staining time depends on thickness of gel.)

1 2 3 4 5 6 7 8 9 10

160

140

120

100

80

60

40

20

Time (hours)

Vol

tage

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4. Following staining, use funnel to decant as much ethidium bromidesolution as possible from staining tray back into storage container. Stainmay be reused to stain 15 or more gels. When staining time increasesmarkedly, disable ethidium bromide solution as explained below.

5. Rinse gel and tray under running tap water to remove excess ethidiumbromide solution. (Chlorine in water will largely inactivate trace amountsof residual ethidium bromide.)

6. If desired, gels can be destained in tap or distilled water for 5 or moreminutes to remove background ethidium bromide.

7. Staining intensifies dramatically if rinsed gels sit overnight. Stack stain-ing trays and cover top gel with plastic wrap to prevent dessication.

8. Wipe down camera, transilluminator, and staining area with 10% bleachsolution.

9. After viewing and photographing, disable stained gels and used stainingsolution:

a. Add 1 volume of 0.05 M KMnO4, and mix carefully.

b. Add 1 volume of 0.25 N HCl, and mix carefully.

c. Let stand at room temperature for several hours.

d. Add 1 volume of 0.25 N NaOH, and mix carefully.

e. Discard disabled solution down sink drain. Drain disabled gels and discard in regular trash.

Viewing and Photographing Gels

Transillumination, in which light passes up through a gel, gives superior viewing of gels stained with either ethidium bromide or Carolina BLU™.

• A fluorescent light box for viewing slides and negatives providesideal illumination for Carolina BLU™-stained gels. An overhead projec-tor may also be used. Cover surface of light box or projector withplastic wrap to keep liquid off the apparatus.

• A mid-wavelength ultraviolet lamp emits in the optimum range for illu-minating ethidium bromide-stained gels (260 to 360 nm). Avoid short-wave lamps, which produce dangerous radiation. Long-wavelength(“black light”) lamps, though safe, give less intense illumination.

Caution: Ultraviolet light can damage the retina of the eye. Neverlook at an unshielded UV light source with the naked eye. Only viewthrough filter or safety glasses that absorb harmful wavelengths.

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• A Polaroid® “gun” camera, equipped with a close-up diopter lens, canbe used to photograph gels on either a UV or white-light transillumina-tor. A plastic hood extending from the front of the camera forms a mini-darkroom and provides correct lens-to-subject distance. Alternatively, aclose-focusing 35-mm camera can be used.

Lab Procedure

Procedure A: Set Up Restriction Digest

1. Students use the four 0.2-mL tubes to perform the restriction reactions:the blue tube contains BamHI, the pink tube contains EcoRI, the greentube contains HindIII, and the yellow tube contains no enzyme.

2. Students should add 20 µL of DNA into each of the colored tubes. Theenzymes in the bottom of the colored tubes contain a dye to aid in visu-alization. Mix the DNA and enzymes by pipetting up and down severaltimes. There should be no concentration of the blue color in the bottomof the tube if the DNA and enzyme are mixed sufficiently. Use a freshtip when adding DNA into each reaction tube to prevent cross contami-nation of enzymes.

3. Incubate all reaction tubes for a minimum of 20 min at 37° C. You mayinstruct the students to incubate the reactions for a longer period.

Procedure B: Cast Agarose Gel

1. Students seal ends of a gel-casting tray and insert a well-forming comb.They should place the gel-casting tray out of the way on lab bench, sothat agarose poured in next step can solidify undisturbed.

2. Students carefully pour enough agarose solution into the casting tray to fillit to a depth of about 5 mm. The gel should cover only about 1⁄3 the heightof comb teeth. While the agarose is still liquid, a pipet tip or toothpick canbe used to move large bubbles or solid debris to sides or end of the tray.

3. The gel will become cloudy as it solidifies (about 20 min). Do not moveor jostle the casting tray while agarose is solidifying.

4. When agarose has solidified, students unseal the ends of the casting trayand place in gel box so that the comb is at the negative (black) end.

5. Students fill box with 1× tris-borate-EDTA (TBE) buffer to a level that justcovers entire surface of the gel.

6. Students must gently remove the comb, being careful not to rip wells.

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7. Make certain that sample wells left by comb are completely sub-merged. If dimples are noticed around wells, students should slowlyadd buffer until the dimples disappear.

8. The gel is now ready to load with DNA. If students will be loading thegel during another period, instruct your students to cover the elec-trophoresis tank to prevent drying of the gel.

Procedure C: Load Gel

1. Students add 2 µL loading dye to each reaction tube, and mix dye withdigested DNA by tapping the tube on lab bench, or with a pulse in amicrocentrifuge.

2. Students use a micropipet to load contents of each reaction tube into aseparate well in the gel. Use a fresh tip for each reaction tube.

• Steady pipet over well using 2 hands.

• Be careful to expel any air in micropipet tip end before loading gel. (Ifan air bubble forms a “cap” over well, the sample will flow intobuffer around edges of well.)

• Dip pipet tip through surface of buffer, position it over the well, andslowly expel the mixture. Sucrose in the loading dye makes the samplemore dense than TBE buffer, causing it to sink to the bottom of thewell. Be careful not to punch tip of pipet through the bottom of the gel.

Procedure D: Electrophorese

1. Students close top of electrophoresis chamber and connect electricalleads to an approved power supply, anode to anode (red-red) and cath-ode to cathode (black-black). Make sure both electrodes are connectedto same channel of power supply.

2. Students turn power supply on and set voltage as directed by you.Shortly after current is applied, loading dye should be seen movingthrough gel toward positive pole of electrophoresis apparatus.

4. Allow the DNA to migrate until the bromophenol blue band from theloading dye nears the end of the gel. You may monitor the progress ofelectrophoresis in the students’ absence; in that case, students omitSteps 5 and 6.

5. Students turn off power supply, disconnect leads from the inputs, andremove top of electrophoresis chamber.

6. Students should carefully remove casting tray and slide gel into stainingtray labeled with their group name. The students should then bring theirgels to you for staining.

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Results and Discussion

Restriction Enzymes

1. Question: Why were the restriction enzyme digests done at 37° C? Answer: Remember that EcoRI enzyme comes from Escherichia coli.This is a bacterium that lives in the intestinal tract of mammals (includ-ing humans). The temperature in the gut is 37° C, so that is the temper-ature at which the enzyme functions. Not all restriction enzymes workat the same temperature; however, 37° C is the temperature at whichmost enzymes work most efficiently.

2. Question: HindIII enzyme comes from Haemophilus influenzae Rd.What does the III in the name refer to? Answer: This was the third restriction enzyme isolated from H. influenzae.

3. Question: Would a BamHI restriction enzyme digest of a different bacte-riophage look the same as the BamHI digest of lambda? Why or whynot?Answer: No, it would not. A restriction enzyme digest of a particulartype of DNA is unique and individual, just like a fingerprint.

Data Analysis

1. Examine your stained gel on a light box or overhead projector. Compareyour gel with the ideal gel shown in Figure 1 (see pg. 26).

2. Question: How can you account for differences in separation and bandintensity between your gel and the ideal gel?Answer: Bands on ideal gel are more spread out, because the gel elec-trophoresed for a longer period of time. Band intensity is dependentupon mass of DNA in the band—the greater the mass of DNA the moreintensely stained the band.

3. Question: Suppose that two small restriction fragments of nearly thesame base-pair size appear as a single band, even when the sample isrun to the very end of the gel. What could be done to resolve the frag-ments? Why would it work?Answer: Increase the concentration of agarose in the gel. The “tighter” gelmatrix more effectively separates smaller DNA fragments. Alternatively,cast a longer gel, and let the DNA electrophorese a longer time.

4. Linear DNA fragments migrate at rates inversely proportional to the log(base 10) of their molecular weights. For simplicity, base-pair length issubstituted for molecular weight.a. The matrix on the next page gives the actual size in base pairs (bp) of

20

BamHI digests, which you will share with the students at Step 4i inthe Data Analysis section of the Student Worksheet).

b. Using the ideal gel shown on page 26, carefully measure distance (in mm) each HindIII, EcoRI, and BamHI fragment migrated from the ori-gin. Measure from front edge of well to front edge of each band. Enterdistances into matrix. Alternatively, have students measure distancedirectly from their Carolina BLU™- or ethidium bromide-stained gel.

HindIII EcoRI BamHI

Dist. Act. bp Dist. Cal. bp Act. bp Dist. Cal. bp Act. bp

*27,491 *24,756 16,841*23,130 *21,226 12,275

9,416 7,412 7,2336,557 *5,804 *6,7704,361 *5,643 *6,5272,322 4,878 *5,6262,027 3,530 *5,505

**564**125

*Pair appears as single band. **Does not appear on this gel.

c. Match base-pair sizes of HindIII fragments with bands that appear inthe ideal digest. Label each band with kilobase-pair (kbp) size. Forexample, 23,130 bp equals 23.1 kbp.

d. Set up semilog graph paper with distance migrated as the x (arith-metic) axis and base-pair length as the y (logarithmic) axis. Then, plotdistance migrated versus base-pair length for each HindIII fragment.

e. Connect data points with a best-fit line.

f. Locate on x-axis the distance migrated by the first EcoRI fragment.Using a ruler, draw a vertical line from this point to its intersectionwith the best-fit data line.

g. Now extend a horizontal line from this point to the y-axis. This givesthe calculated base-pair size of this EcoRI fragment.

h. Repeat Steps f and g for each EcoRI and BamHI fragment. Enterresults in the calculated base pairs (Cal. bp) columns for each digest.

i. Enter the actual base-pair size of EcoRI and BamHI fragments, asprovided by your instructor, into Act. bp columns.

j. For which fragment sizes was your graph most accurate? For which

fragment sizes was it least accurate? What does this tell you about theresolving ability of agarose-gel electrophoresis?

Answer: Extrapolations from the graph are most accurate for short tomid-size DNA fragments and least accurate for very large fragments.The 0.8% gel used in this experiment most effectively separates shortto mid-size fragments.

21

Field Guide to Electrophoresis Effects

B E H — B E H — B E H —

B E H —

B E H —

B E H —

Ideal Gel

Short Run

Bands compressed. Shorttime electrophoresing.

Overload

Bands smeared in alllanes. Too much DNAin digests.

Punctured Wells

Bands faint in lanes B and H. DNA lost throughhole punched in bottomof well with pipet tip.

Long Run

Bands spread. Longtime electrophoresing.

Underloaded

Bands faint in alllanes. Too little DNAin digests.

22

23

B E H —

Poorly Formed Wells

Wavy bands in all lanes.Comb removed beforegel was completely set.

B E H — B E H —

B E H — B E H — B E H —

Enzymes Mixed

Extra bands in lane H.BamHI and HindIIImixed in digest.

Precipitate

Precipitate in TBE bufferused to make gel.

Bubble in Lane

Bump in band in lane B.Bubble in lane.

Incomplete Digest

Bands faint in lane H.Very little HindIII indigest. Also, extrabands are present inlanes B and E.

Gel Made withWater

Bands smeared in alllanes. Gel made withwater or wrong concen-tration of TBE buffer.

IntroductionIn this experiment, DNA from the bacteriophage lambda (48,502 base pairs in length) is cut with restrictionenzymes and the resulting restriction fragments are separated using gel electrophoresis. Three samples of lambdaDNA are incubated at 37° C, each with 1 of 3 restriction endonucleases: BamHI, EcoRI, and HindIII. A fourthsample, the negative control, is incubated without an endonuclease.

The DNA samples are then loaded into wells of an agarose gel and electrophoresed. An electric field appliedacross the gel causes the DNA fragments in the samples to move from their origins (sample wells) through the gelmatrix toward the positive electrode. Smaller DNA fragments migrate faster than larger ones, so restrictionfragments of differing sizes become concentrated into separate bands during electrophoresis. The characteristicnumber and pattern of bands produced by each restriction enzyme are, in effect, a “DNA fingerprint.” Therestriction patterns are made visible by staining with a compound that binds to DNA.

Procedure

A: Set Up Restriction Digest

1. You will receive 4 reaction tubes. Three of these tubes contain the restriction enzymes and the fourth tubecontains no enzyme and is a negative control. The reaction tubes are color coded as follows:

EcoRI Pink TubeBamHI Blue TubeHindIII Green TubeNo Enzyme Yellow Tube

The enzymes contain a dye to aid in visualization and are in dehydrated form in the bottom of each tube.

2. Add 20 μL of DNA to one of the tubes. Mix the dried enzyme with the DNA by pipetting the DNA up anddown a few times. Place the tube upright in the provided rack.

3. Repeat the process for each enzyme tube and the yellow control tube, using a fresh pipet tip between eachtube to prevent cross contamination between the tubes.

4. Cap each tube with the matching colored lid.

5. Incubate all reaction tubes for a minimum of 20 min at 37° C. Your teacher may instruct you to incubate thereactions for a longer period.

B: Cast Agarose Gel

1. Seal ends of gel-casting tray and insert well-forming comb. Place gel-casting tray out of the way on lab bench,so that agarose poured in next step can solidify undisturbed.

2. Carefully pour enough agarose solution into casting tray to fill to depth of about 5 mm. Gel should cover onlyabout 1⁄3 the height of comb teeth. Use a pipet tip or toothpick to move large bubbles or solid debris to sides orend of tray while gel is still liquid.

3. Gel will become cloudy as it solidifies (about 20 min). Do not move or jar casting tray while agarose is solidifying.

4. When agarose has solidified, unseal ends of casting tray. Place tray in gel box so that comb is at negative(black) end.

5. Fill box with 1× tris-borate-EDTA (TBE) buffer, to a level that just covers entire surface of gel.

Student Instructions Name

21-1185, 21-1187 Date

Restriction Enzymes and DNA Kit

24

6. Gently remove comb, being careful not to rip wells.

7. Make certain that sample wells left by comb are completely submerged. If “dimples” are noticed aroundwells, slowly add buffer until they disappear.

8. The gel is now ready to load with DNA. If you will be loading the gel during another period, your teacher willinstruct you to cover the electrophoresis tank to prevent drying of the gel.

C: Load Gel

1. Add 2 µL loading dye to each reaction tube. Mix dye with digested DNA by tapping tube on lab bench or witha pulse in microcentrifuge.

2. Use micropipet to load contents of each reaction tube into a separate well in gel, aligned as illustrated in theideal gel in Figure 1. Use a fresh tip for each reaction tube.

a. Steady the pipet over well using two hands.

b. Be careful to expel any air in micropipet tip end before loading gel. (If air bubble forms “cap” over well,DNA and loading dye will flow into buffer around edges of well.)

c. Dip pipet tip through surface of buffer, position it over the well, and slowly expel the mixture. Sucrose inthe loading dye weighs down the sample, causing it to sink to the bottom of the well. Be careful not topunch tip of pipet through bottom of gel.

D: Electrophorese

1. Close top of electrophoresis chamber and connect electrical leads to an approved power supply, anode toanode (red-red) and cathode to cathode (black-black). Make sure both electrodes are connected to samechannel of power supply.

2. Turn power supply on and set voltage as directed by your instructor. Shortly after current is applied, loadingdye should be seen moving through gel toward positive pole of electrophoresis apparatus.

3. The bromophenol blue in the loading dye migrates through gel at the same rate as a DNA fragmentapproximately 300 base pairs long.

4. Allow the DNA to migrate until the bromophenol blue band nears the end of the gel. Your instructor maymonitor the progress of electrophoresis in your absence; in that case, omit Steps 5 and 6.

5. Turn off power supply, disconnect leads from the inputs, and remove top of electrophoresis chamber.

6. Carefully remove casting tray and slide gel into staining tray labeled with your group name. Take gel to yourinstructor for staining.

Results and Discussion

Restriction Enzymes

1. Why were the restriction enzyme digests done at 37° C?

2. HindIII enzyme comes from Haemophilus influenzae Rd. What does the III in the name refer to?

3. Would a BamHI restriction enzyme digest of a different bacteriophage look the same as the BamHI digest oflambda? Why or why not?

Data Analysis

1. Examine your stained gel on a light box or overhead projector. Compare your gel with the ideal gel shown inFigure 1.

2. How can you account for differences in band separation and intensity between your gel and the ideal gel?

25

3. Suppose that two small restriction fragments of nearly the same base-pair size appear as a single band, evenwhen the sample is run to the very end of the gel. What could be done to resolve the fragments? Why wouldit work?

4. Linear DNA fragments migrate at rates inversely proportional to the log10 of their molecular weights. Forsimplicity, base-pair length is substituted for molecular weight.

a. The matrix below gives the actual size in base pairs (Act. bp) of lambda DNA fragments generated by aHindIII digest:

HindIII EcoRI BamHIDist. Act. bp Dist. Cal. bp Act. bp Dist. Cal. bp Act. bp

*27,491*23,130

9,4166,5574,3612,3222,027**564**125

*Pair appears as single band. **Does not appear on this gel.

Figure 1 Ideal gel

BamHI EcoRI HindIII No Enzyme

26

Carolina Biological Supply CompanyCB270329803

2700 York Road, Burlington, North Carolina 27215

b. Using the ideal gel shown in Figure 1, carefully measure distance (in mm) each HindIII, EcoRI, and BamHIfragment migrated from the origin. Measure from front edge of well to front edge of each band. Enterdistances into matrix.

c. Match base-pair sizes of HindIII fragments with bands that appear in the ideal digest. Label each band withkilobase-pair (kbp) size. For example, 23,130 bp equals 23.1 kbp.

d. Set up semilog graph paper with distance migrated as the x (arithmetic) axis and base-pair length as the y(logarithmic) axis. Then, plot distance migrated versus base-pair length for each HindIII fragment.

e. Connect data points with a best fit line.

f. Locate on x axis the distance migrated by the first EcoRI fragment. Using a ruler, draw a vertical line fromthis point to its intersection with the best-fit data line.

g. Now extend a horizontal line from this point to the y axis. This gives the calculated base-pair size of this EcoRIfragment.

h. Repeat Steps f and g for each EcoRI and BamHI fragment. Enter results in the calculated base-pairs (Cal. bp)columns for each digest.

i. Enter the actual base-pair size of EcoRI and BamHI fragments, as provided by your instructor, into Act. bpcolumn.

j. For which fragment sizes was your graph most accurate? For which fragment sizes was it least accurate?What does this tell you about the resolving ability of agarose gel electrophoresis?

27

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CB270319803

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