lesson module ii – marker assisted selection iilaboratory lesson plan: agarose gel analysis of the...

117Iowa State University Extension and ISU Office of Biotechnology

EducatorsLesson Module II – Marker Assisted Selection

Marker Assisted

Selection

Part II

TEACHING RESOURCES

Laboratory Lesson Plan:

Agarose Gel Analysis of the

K-Casein B Allele

II

Introduction to K-Casein Proteins

A decade ago, milk produced by cows in the WesternWorld had a lower fat to protein ratio than consumerswanted. This excess milk protein was disposed of onworld markets in the form of dried skim milk. Today'strend in dairy production is to focus on more produc-tion of milk protein. As pricing systems increasinglyinclude payment for the amount of protein in themilk, breeding decisions have focused on proteinproduction. Two protein genes have been identifiedthat affect milk protein production. It is now relativelysimple to determine the genotypes of cows for theseprotein genes.

Studies conducted in the early 1970s indicated that thegene that controls the production of K-casein had thegreatest effect on quality and quantity of cheeseproduction. Two alleles of the K-casein gene have beenidentified as the A and B alleles. Cattle carry two copiesof the A allele, denoted AA, or two copies of the B allele,BB, or one copy of each, AB. The B allele causes asubstantial increase of milk protein yield in the cowscarrying it. The B protein of K-casein results in a firmercheese and enhanced cheese yield from milk. Thegenotype BB yields more protein than AB, which yieldsmore than AA.

Since the frequency of the K-casein B allele in dairybreeds is low (20%), it would be worthwhile to havebreeding programs that emphasize increasing thefrequency of the B allele in the dairy cattle population.Some countries have called for national breedingprograms for increasing the frequency of the B allele intheir dairy herds. Because of AI (artificial insemination)technology, a bull's genotype is of particular interest.

In this activity, an Iowa dairy farmer wants to takeadvantage of the premium pricing for milk thatcontains more K-casein protein that cheese producersvalue. The farmer has enough money to buy one bulland two cows. The farmer also hopes to recover someof the initial cost by selling the bull's semen to otherdairy farmers through an AI program.

Students will analyze the PCR products using restric-tion enzymes and agarose gel electrophoresis todetermine the best bull and cows to purchase andadvise the farmer of the best breeding options toincrease the overall milk protein production inthe herd.

Science Education Standards

Science as Inquiry, Content Standard A

– Abilities necessary to do scientific inquiry(p. 175)

– Understanding about scientific inquiry (p. 176)

Life Science, Content Standard C

– The cell (p. 184)– Molecular basis of heredity (p. 185)– Matter, energy, and organization in living systems

(p. 186)

Science and Technology, Content Standard E

– Understandings about science and technology(p. 192)

Source: National Science Education Standards, ©National Academy ofSciences, 1996. Used with permission. Page numbers refer to theseventh printing, November 1999 – also available on the Internet athttp://books.nap.edu/html/nses/pdf/index.html.

Science Process Skills

• Comparing and measuring• Observing• Ordering• Relating• Inferring

Life Skills

• Learning to learn• Science processing• Problem solving• Decision making• Communicating

118 Iowa State University Extension and ISU Office of Biotechnology

Educators Lesson Module II – Marker Assisted Selection

TimePreparation: Two to three hours, depending on thelevel of student involvement in the preparationsActivity: Three to four 40-minute blocks of class time.

Materials

• Six boxes of pipette tips (already autoclaved)

• 50 μl of 0.030 μg/μl of pKAN DNA (minimum of100 μl per school)

• 40 μl of 0.025 μg/μl pUC19 DNA per class section(minimum of 80 μl per school)

• 3 μl Bgl 1 per class section (minimum of 6 μl perschool)

• 15 μl reaction buffer per class section (minimum of30 μl per school)

• 144 μl Carolina Blu Gel Stain per gel (minimum oftwo gels)

• 732 μl Carolina Blu Buffer Stain per gel (minimumof two gels)

• One bottle Carolina Blu DNA Stain

• 0.9 g agarose per gel (minimum of two gels)

• 70 ml 10X TBE per gel (minimum of two gels) tomake 1X TBE buffer solution for each gel

• 40 μl blue migration dye for each group of fivestudents in all sections.

• Eight microcentrifuge tubes for each group of fivestudents in all sections (already autoclaved)

• K-casein B variant background information andfingerprinting instruction handouts MAS-4through MAS-10 on p. 131-145 and overheadtransparency masters MAS n-p on p. 147-151.


K-Casein B AlleleInstructions for Use of a Pipettor

Prepared by the Office of Biotechnology, Iowa StateUniversity

To conduct the analysis of the K-casein B allele,students will need to know how to use micropipettors.

Teachers can download the step-by-step instructions forthe following procedures/protocols at:www.biotech.iastate.edu/publications/pt_ presentations/default.html.

The following description is of the Labnet pipettorprovided by the Office of Biotechnology. The instru-ment is designed to accurately measure volumes ofliquid from 2 to 20 μl. Before using a pipettor, pleaseread these instructions. Use water to practice loadingand transferring liquids.

Purpose of a Pipettor

Biotechnologists use a pipettor to transfer preciseamounts of liquid from one container to another. Thevolume of liquid transferred generally is extremelysmall and is measured in microliters (μl). There are1,000 μl in 1 ml.

Care of the Pipettor

A pipettor is an expensive and delicate instrument thatmust be used with care. Misuse of the instrument cancause it to measure the incorrect volume of liquid or, inextreme circumstances, fail to measure any volume.The two most important factors to consider are:

1. Do not drop the pipettor.

2. For the pipettor furnished by the Office ofBiotechnology, do not adjust the volume toless than 2 μl or greater than 20 μl anddo not twist the yellow pushbutton.

Parts of the Pipettor

The primary parts of the pipettor are a yellowpushbutton or plunger (1) that is used to measure thedesired volume, a calibration knob (2) that is used toset the pipettor for the volume of liquid to be measured,a digital display (3) of thevolume that the pipettor isset to measure, the pipettebarrow (4) and the pipettecone (5) on which adisposable pipette tip isplaced to hold the liquid.Another important part ofthe pipettor is thepushbutton ejector (6) thatis used to remove a usedpipette tip from the pipettecone (5).

Figure 1



Figure 2

Adjustment of the Volume

The digital display (3) in the handle of the pipettorconsists of three vertical numbers. The upper twonumbers indicate μl and the bottom number indicates0.1 μl. The red line between the top two numbers andthe bottom number represents the decimal point. Asetting of 070 is 7.0 μl , a setting of 075 is 7.5 μl , and asetting of 170 is 17.0 μl. The pipettor provided bythe Office of Biotechnology is designed to accuratelymeasure volumes between 020 (2.0 μl) and 200(20.0 μl).

To set the pipettor for the desired volume, turn thecalibration knob so that the digits increase or decrease,as required. The pipettor will be damaged if thecalibration knob is forced to turn at settings below 020or above 200.

Method for Transferring Liquid

1. Adjust the pipettor to the desired volume.

2. Hold the pipettor in the hand so that the digitaldisplay is facing the person using it and the thumbcan easily depress the yellow pushbutton.

3. Open the box containing the pipette tips. Placethe pipette cone into the pipette tip and pressdown firmly until the tip remains on the cone.Use of excess pressure can damage the pipettor.Close the cover of the box after the tip has beenremoved to minimize contamination of theremaining tips in the box. After the pipette tip isattached, do not let the tip come into contactwith any object because the tip will becomecontaminated.

NEVER USE THE PIPETTOR WITHOUT A TIPATTACHED because the entire instrument willbe contaminated. If contamination accidentallyoccurs, rinse the pipettor thoroughly by loadingand unloading 20.0 μl volumes of distilled water.

4. Before the pipette tip is placed in the liquid to betransferred, press the yellow pushbutton/plungerto the first stop that can be felt (A). See Figure 2below.

If the button is pushed as far down as it will go,the desired volume will not be measured. This isreferred to as “over pipetting.” If you over pipette,you will draw the entire contents of the tube intothe tip and you will not have enough reagent tocomplete the activity. If the button is depressedwhile it is in the liquid to be transferred, bubbleswill be produced, which can make it difficult toget liquid into the pipette tip.

With the button depressed to the first stop, placethe pipette tip into the liquid to be transferred (B).Slowly release the button while keeping thetip in the liquid (C). After the button is fullyreleased, remove the tip of the pipettor from theliquid. Do not wipe the tip or let it come intocontact with any surface. Keep the pipettorvertical at all times.

5. Place the tip of the pipettor into the container towhich the liquid is being transferred (D). If thecontainer already contains liquid, place the tipinto the liquid. Slowly press the yellow push-button down as far as it will go to eject all the



liquid from the tip (E). Keep the button fullydepressed as you remove the tip of the pipettorfrom the container.

When the liquid being transferred is being addedto another liquid, it may be desirable to mix thetwo. Place the pipette tip containing the liquidbeing transferred into the second liquid. Slowlypress the button down as far as it will go (E).Without removing the tip, slowly release thebutton to draw liquid back into the tip (C).Slowly depress the button down as far as it willgo (E). Repeat the process several times. Thelast time, depress the button as far as it will go(E) and remove the pipettor’s tip from thecontainer before releasing the button.

6. Remove the used pipette tip by pressing down onthe ejector button (F) or pulling the tip off byhand. Dispose of the tip in an appropriatecontainer.

Practice Gels

At this point, it can be beneficial to prepare practice gelsto help teach the transfer of liquids into the wells of theagarose gel. To make practice gels, melt Knox® gelatinin hot water and either pour the liquid into the castingtrays of the gel box or into Petri dishes. Add the combbefore the gelatin hardens. After 20 minutes, pull thecombs out to expose the wells. Depending on the sizeof the wells you use, you can load between 10-20 μl ofcake dye into the wells. Cake dye is preferred becauseof its higher density than food coloring. Several groupscan share a gel.Knox® is a registered trademark of NBTY, Inc., used by Kraft Foodsunder license.

PowerPoint® TutorialA PowerPoint® tutorial about using a micropipettor isavailable for downloading (4Mb size) on the WorldWide Web at www.biotech.iastate.edu/publications/ppt_presentations/default.html. There is also an html (Webpage) version of the tutorial at this site.

In addition, the tutorial has been placed on a compactdisc (CD) that can be opened on either PC or Mac-intosh computers with CD or DVD drives. The CD ofeducational resources from Iowa State University’sOffice of Biotechnology is provided free to Iowateachers. To order the CD, phone 515-294-9818, toll-free in Iowa 800-643-9504, or e-mail [email protected]® is a registered trademark of Microsoft Corporation.


K-Casein B Allele

Prepared by the Office of Biotechnology, Iowa StateUniversity

The preparation and conduct of the genotypinglaboratory is divided into the following sections:

I. Preparation of the student materialsII. Plasmid DNA preparationIII. Restriction endonuclease preparationIV. Migration dye preparationV. Preparing, loading and running an agarose gel

for use with Carolina Blu Stain

I. PREPARATION OF THE STUDENT MATERIALS

The supplies can best be provided to the class in groupsof five students. The DNA samples should be kept inthe refrigerator until the class is set up. It is tooexpensive for the Office of Biotechnology to provideDNA for every student in a class. It will provideenough DNA, restriction endonuclease, and reactionbuffer for a minimum of two groups of five students, orit will provide enough for one group of five students inevery class section, whichever is greater. For theremaining groups of students, use distilled water toreplace the DNA, restriction endonuclease, and reactionbuffer. Every group of students should be providedwith the blue migration dye.

For each set of DNA, you will receive two differenttubes of DNA. In each set there will be enough DNAand enzyme to do two groups. A set will contain:100 μl of 0.030 μg/μl of pKAN, 80 μl of 0.025 μg/μl ofpUC19, 50 μl of restriction buffer and 9 μl ofBgl 1 enzyme.

Prepare for the lab in the following manner:

• Label seven 1.5 ml microcentrifuge tubes M1, M2,F1, F2, F3, N, and D. The label should be writtenon both the cap and side of the tube with apermanent felt pen.

• In tube M1, add 8.5 μl of pKAN DNA. With afresh tip add 8.5 μl of pUC19 to the tube.

• In tube M2, add 17 μl of pKAN DNA.

• In tube F1, add 17 μl of pUC19 DNA.

• In tube F2, add 8.5 μl of pKAN DNA. With a freshtip add 8.5 μl of pUC19 to the tube.



• In tube F3, add 17 μl of pKAN DNA.

• One microcentrifuge tube (1.5 ml) containing 18μl of a mixture of 3 μl Bgl 1 and 15 μl of reactionbuffer. The tube should be labeled “N.”

• One microcentrifuge tube (1.5 ml) containing 40μl of blue migration dye and labeled “D”

• One 20 μl pipettor per group

• 10 sterile pipette tips of 200 μl in an appropriatecontainer per group

• One container to hold the used pipette tips

• 144 μl Carolina Blu gel stain per gel

• 732 μl of Carolina Blu buffer stain per gel

• One bottle of Carolina Blu final stain per two gels

• 0.9 grams of agarose per gel

• 70 ml of 10X TBE mixed with 630 ml of distilledwater to make 700 ml of 1X TBE buffer solutionper gel

• Five copies of the laboratory instructions MAS-5through MAS-10 on p. 133-145 (one for eachgroup of students)

• Each group should be assigned a letter (A,B,C,D, orE) and write it in the upper right hand corner ofthe instruction sheets.

The teacher should have available for the entire class:

• Electrophoresis gel• Electrophoresis power supply• One 20 μl pipettor and 1 box of sterile 200 μl

pipette tips for each gel box• An incubator at 37º C and rack to hold the

microcentrifuge tubes of the students• One Sharpie® marking pen• One sheet of paper for each gel box that has num-

bered lines or boxes corresponding to the lanes onthe gel

• (Optional) Enough small, medium, large, andextra large medical gloves for the students

Teachers in Iowa can contact the Office of Biotechnol-ogy, Iowa State University, 1210 Molecular BiologyBuilding, Ames, IA 50011

ph: 515-294-9818 or 800-643-9504fax: 515-294-4629

List of biological supply companies for teachers notin Iowa:

Carolina Biological Supply Company800-334-5551www.carolina.com/

Fisher Science Education800-955-1177www1.fishersci.com/education/

MBI Fermentas800-340-9026www.fermentas.com/

Supplies Provided to Iowa Teachers by the Office ofBiotechnology, Iowa State University

Iowa teachers should request the following supplies atleast a week in advance. Supplies should be orderedby contacting Lori Miller by phone, 800-643-9504,or 515-294-9818, or by fax: 515-294-4629. Theelectrophoresis equipment should be obtained bycontacting the Area Education Agency or the ISUOffice of Biotechnology.

• Six boxes of pipette tips (already autoclaved)

• 50 μl of 0.030 μg /μl pKAN per class section(minimum of 100 μl per school)

• 40 μl of 0.025 μg /μl pUC19 DNA per classsection (minimum of 80 μl per school)

• Three μl Bgl 1 per class section (minimum of 9 μlper school)

• 15 μl reaction buffer per class section (minimumof 50 μl per school)

• 144 μl Carolina Blu Gel Stain per gel (minimum of2 gels)

• 732 μl Carolina Blu Buffer Stain per gel (minimumof 2 gels)

• One bottle Carolina Blu DNA Stain

• 0.9 g agarose per gel (minimum of 2 gels)

• 70 ml 10X TBE per gel (minimum of 2 gels)



• 40 μl blue migration dye for each group of fivestudents in all sections.

• Eight microcentrifuge tubes for each group of fivestudents in all sections (already autoclaved)

II. PLASMID DNA PREPARATION

It is best if medical or dishwashing gloves are wornwhen preparing supplies for the laboratory to preventthe teacher’s fingers from contaminating the DNAsamples. The DNA will not harm the teacher, but theteacher can harm the DNA.

The DNA samples are prepared from plasmid DNAprovided at the appropriate concentration by the Officeof Biotechnology for Iowa teachers. Teachers not inIowa and/or those choosing to get their own DNA candilute their DNA by using the following formula:

C1 x V

1 = C

2 x V

2

C1 is the initial DNA concentration from the supplier

(listed as μg /μl). V1 is the volume of DNA you calcu-

late you need for your final solution. C2 is the concen-

tration of DNA you need for the activity. V2 is the

volume of the solution you have to make to completethe activity.

For the pKAN DNA solution, you need 100 μl of 0.030μg /μl. The easiest way to calculate the amount ofconcentrated DNA you will need is to take 100 μl x0.030 μg /μl and divide that product by the concentra-tion (μg /μl ) from the supplier. You mix that volume ofconcentrated DNA with enough sterile distilled water orTE buffer to reach your total desired volume.(Example: 6 μl of concentrated DNA in 94 μl of steriledistilled water equals 100 μl of DNA solution forthe activity.) The DNA should be kept refrigerated,except when it is being used to prepare and conductthe laboratory.

A final volume 17 μl of the plasmid DNA from theappropriate supply tubes should be put into sterile 1.5ml microcentrifuge tubes for the students. The tubescan be prepared up to 24 hours in advance and kept ina refrigerator until the class period. The number oftubes of each type for each group of five students isdescribed in Section I, Preparation of the StudentMaterials, beginning on p. 120.

III. RESTRICTION ENDONUCLEASEPREPARATION

The restriction endonuclease used for the laboratory isBgl 1, commonly referred to as Bagel 1. The endonu-

clease and the reaction buffer can be prepared for thestudents up to 24 hours in advance and kept in arefrigerator until the class period. The mixture shouldbe prepared in separate tubes for each group of stu-dents, ie. a large amount should not be prepared andsubdivided into different tubes.

To obtain 18 μl of the mixture for a group of fivestudents, 15 μl of the reaction buffer is added to a sterile1.5 microcentrifuge tube and then 3 μl of Bgl 1 is addedto the tube. To rinse the pipette tip and mix the Bgl 1and reaction buffer, fill and unload by pushing downand releasing the pipette’s plunger three times. Thetube should be clearly labeled as “N.”

IV. MIGRATION DYE PREPARATION

A blue migration dye is used to monitor the movementof the DNA during electrophoresis. The migration dyeis provided by the Office of Biotechnology to Iowateachers ready for use. Each group of students shouldreceive the dye, even if they are given placebos ofwater, instead of DNA, restriction endonuclease, andreaction buffer.

V. PREPARING, LOADING, AND RUNNING ANAGAROSE GEL FOR USE WITH CAROLINABLU STAIN

The gel can be prepared up to two days in advance ofthe period in which the DNA is loaded into it. Ifprepared in advance, 1) place the electrophoresis box inthe refrigerator with the cover in place or 2) remove thecasting tray and gel from the electrophoresis box, seal ina plastic bag, and store in a refrigerator.

The following description applies to the 12 cm wide x14 cm long electrophoresis unit provided by the Officeof Biotechnology, Iowa State University. The generalprocedures would be the same for any gel apparatus,except for the volumes of gel and buffer that are used.

1. Put on a pair of medical or dishwashing glovesand wear them throughout the procedure,including during the laboratory and the clean up.None of the chemicals used are toxic, but thegloves provide protection for persons who mayhave sensitive skin.

2. The electrophoresis box will come completelyassembled. To prepare the box for casting a gel,follow these directions:



a. To remove the lid from the box, face the boxwith the electrode plugs pointing to the back,and place fingers on each end of the unit whilepressing thumbs against the front edge of thelid. Push thumbs against the lid toward therear to disconnect the power supply leads fromthe plugs. Lift lid to remove from system.

b. Remove the gel tray by grasping each side and lifting at an angle to ease the tray out ofthe system (See Figure 3). Rinse the combs,gel tray and gel box in distilled water toremove any residue. It is not necessary to drythe pieces.

c. Replace the gel tray in the system by carefullylowering the tray at an angle so the gaskets fitagainst the front and rear of the gel box wall(see Figure 3). This should provide anefficient seal that prevents leakage of the warmagarose when poured into the tray. If thegasket has slipped out of the groove, push itback in place before lowering the tray intothe box.

3. Prepare 700 ml of 1X TBE electrophoresis bufferby diluting 70 ml of 10X TBE stock solution with630 ml of distilled water.

4. Pour into a 250 ml flask or beaker 45 ml of 1XTBE electrophoresis buffer prepared in step 3 andadd 0.9 g of agarose. Swirl the agarose suspen-sion to disperse the powder.

5. Put on a heat-resistant glove. Microwave thesuspension until it boils (about 30 seconds to 1minute), swirl the flask, and alternate boilingand swirling at 15 second intervals until thesolution has boiled a total of 1 minute or untilthere are no visible solids.

6. Cool the agarose solution by adding another 45ml of the 1X TBE buffer from step 3, bringing the

volume in the container to 90 ml. Swirl thesolution gently to avoid trapping air bubbles.

7. While the agarose solution is still liquid, add144 μl of Carolina Blu Gel Stain to the agarosesolution. The Gel Stain will be provided fromthe Office of Biotechnology in a microcentrifugetube for Iowa teachers. To get all the stain out ofthe tube, rinse the tube with 1X TBE buffer ordistilled water and pour it in the agarose solu-tion. Swirl the agarose solution gently until ithas a uniform light blue color.

8. Slowly pour the agarose solution into the gel tray,taking care not to allow formation of any bubbleswithin the gel. If bubbles form, tap them with afinger until they disappear.

9. Rinse the flask or beaker immediately withplenty of tap water to prevent the agarose fromhardening in it or the sink.

10. Immediately place a comb(s) of choice into thetray slot, attempting to avoid air bubble forma-tion in the wells. Allow the gel to sit at roomtemperature for about 30 minutes until solid(gel will appear slightly milky).

11. After the gel is set, remove the comb(s). Toremove a comb, grasp both ends of the comb andgently lift straight up with a slight back and forthrocking motion.

To orient the gel tray in the running position,grasp the sides of the tray and gently lift at anangle (see Figure 3).

Rotate the tray 90 degrees to position the openends toward the platinum electrodes. DNA isnegatively charged and will migrate toward thepositive (red) pole during electrophoresis. Thewells of the gel should be nearest the negativeelectrode (black) end of the electrophoresis box.

Carefully lower the tray into position, and securethe tray between the gel tray tabs. To verify thatthe gel tray is properly oriented, place the lidloosely on the box. The wells should be nearestthe negative electrode (black).

12. Load the DNA samples by placing the pipette tipinto the top of the well and slowly release thesolution into the well (See Figure 4). Thepipettor can be kept steady by holding the barrel

Figure 3



like a pool stick and leaning on the gel box. Putthe tip into the well as vertically as possible. Donot go too deep into the well to avoid puncturingthe gel.

13. After the students have loaded their samples intothe wells, add 732 μl of Carolina Blu buffer stainto the 610 ml of 1X TBE buffer that remainedafter step 6. For Iowa teachers the buffer stainwill be provided from the Office of Biotechnol-ogy in a microcentrifuge tube. To get all thestain out of the tube, rinse the tube with distilledwater and pour it into the buffer. Swirl the bufferuntil it is a uniform light blue color.

14. Slowly fill the gel box with all the 1X TBEelectrophoresis buffer to cover the gel to about a2 mm depth. Do not pour the buffer directlyon the gel.

15. Make sure the switch on the power supply is inthe “Off” position before connecting the electro-phoresis chamber. When ready for electrophore-sis, place the lid tightly on the chamber and plugthe electrical leads into the recessed output jacksof the power supply. Plug the red(+) lead intothe red jack, and the black(-) lead into theblack jack.

16. For operation of the power supply, follow theinstructions provided with it.

17. Select the desired voltage on the power supply. Avoltage of 150 will permit the electrophoresis runto be completed in about an hour. Lowervoltages also can be used. The lower the voltage,the slower the DNA will migrate. For example,at a voltage of 10, the electrophoresis run will becompleted in about 24 hours. The band ofmigration dye marks the leading edge of theDNA. The electrophoresis is complete when theleading edge of the dye has migrated half waydown the gel.

18. Proceed with electrophoresis: Check to be surethe blue migration dye is moving toward thepositive electrode (red). If it is migrating towardthe negative electrode (black), turn off the powersupply and switch the position of the electricalleads to the power supply and repeat steps 16and 17. CAUTION: Never remove the electro-phoresis chamber lid or change the positionto the leads while the power supply is on.

19. When electrophoresis is completed, turn off thepower supply.

20. To remove the lid from the box, face the boxwith the electrode plugs pointing to the back,and place fingers on each end of the unit whilepressing thumbs against the front edge of the lid.Push thumbs against the lid toward the rear todisconnect the power supply leads from theplugs. Lift the lid to remove it from the box.

21. After the electrophoresis is complete, some DNAbands will be visible. To darken the bands andmake more of them visible, remove the gel trayfrom the electrophoresis unit and place the trayinto a plastic container. Slide the gel off of thetray by pushing on one end of it. Add theCarolina Blu DNA Final Stain, making sure thegel is completely immersed. Do not pour thestain directly on the gel.

22. Stain the gel for 15 minutes. Agitate gently, ifpossible.

NOTE: If the final stain has been used morethan once, add 5 to 10 minutes to the stainingtime. A total of six gels can stained with onebottle of final stain.

23. Pour the stain back into the bottle.

24. Cover the gel with distilled water to destain.(Tap water contains chloride ions that canpartially remove the stain from the DNA bandsand give inferior results.) Agitate gently, ifpossible. During the 30 to 40 minutes ofdestaining, change the water every 10 minutes, ifpossible. During the destaining process, thebands of DNA will become clearer as thestain is removed from the remainder of the gel.It is possible to destain the gel for up to 24 hoursby adding enough distilled water to barely coverthe bottom of the tray (do not flood the gelovernight) and covering it with aluminum foil.If the DNA bands become too light, the gel can

Correct Incorrect

Figure 4

Incorrect



be stained and destained again by repeating steps21 through 24.

25. The gel can be displayed to the class by sliding itinto a sealed plastic bag with a small amount ofdistilled water. The gel can be viewed on a whitesheet of paper or a white-light viewing boxwithout removing it from the bag. Figure 5illustrates the expected results.

26. The gel can be saved for at least a month in arefrigerator by sliding it into a clear plastic bagand sealing the bag.

27. None of the chemicals used in the experimentare toxic. Solutions can be poured down aconventional drain. The gel can be disposed ofwith other trash.

Alternative Staining Procedure

An alternative staining procedure allows teachers tostain the gel overnight. Follow steps 1-20 above.Carefully place the gel into a one-gallon, top sealing(ziplock) bag. Make a 50-50 mixture of final stain anddistilled water and pour that into the bag with the gel.Seal and let stain overnight. If you choose to destain,drain the stain into a container and save it so it can beused again. Add distilled water to the bag, seal, anddestain to the desired intensity. By diluting the finalstain to 25%, you can avoid the destaining step. If youwant the DNA bands on the gel to be darker, add morefinal stain to the bag and let it sit until the desiredintensity is reached.

WARNING: Be very careful in handling the gel in thebag. The flexibility of the bag allows the gel to bendand break easily. When handling the gel in the bag, be

sure to support the gel with your hand. Place the gelflat on a table top during staining and destaining.

FITTING THE GENOTYPING EXPERIMENT INTOTHREE 45-MINUTE PERIODS

Period 1:Teach how to use the pipettor and make the agarose gel.After the gel is made, the teacher has two options:

a. Place the electrophoresis box in the refrigeratorwith the cover in place until period 2 or

b. The casting tray and gel can be removed from theelectrophoresis box, sealed in a plastic bag, andstored in a refrigerator until period 2. The gelcan be kept in the refrigerator for up to 2 daysbefore it is used.

Period 2:Remove the container with the gel from the refrigerator.Conduct steps 1 (optional) through 6 of the studentinstructions on p. 133-134. In step 3, the incubation at37º C can be done for up to 45 minutes, if desired bythe teacher. If the incubated DNA is not going to beloaded by the students into the gel immediately, it canbe stored in a refrigerator or freezer until the migrationdye is added and the gel is loaded.

After the DNA is loaded into the gel by the students, theteacher has three options:

a. Carry out the electrophoresis or

b. If students in another section are to load DNAinto the same gel on the same day, place thecover on the electrophoresis box and store it in arefrigerator until the next class period. After thegel is loaded by the last section, add the electro-phoresis buffer and carry out the electrophoresis

orc. If another gel is to be loaded on the same day by

another class section, place the cover on theelectrophoresis box with the loaded gel and storeit in a refrigerator until both gels can be run.After both gels are ready, add the electrophoresisbuffer and carry out the electrophoresis.

After the electrophoresis is completed, there are twooptions for staining:

a. Stain the gel immediately and place it in therefrigerator, in a clear, sealed plastic bag or

Figure 5 – Genotype Key for K-Casein B

AA AB BB



b. Pour enough of the electrophoresis buffer out ofthe electrophoresis box so that the gel is notimmersed in it. Place the cover on the box andstore it in the refrigerator for up to a day until itis convenient to stain it.

Period 3:Stain the gel (if it has not already been done), view thegel, and discuss the results.

Agarose Gel Analysis of the K-Casein B

Allele: Procedure on the Day of the

Laboratory

Divide the class into groups of five students each. Usehandouts MAS-4 and -5 on p. 131-135 to present thefollowing “Which Cattle Should I Buy?” scenario orverbally tell the students:

“A farmer in Iowa is interested in increasing the proteincontent in the milk of the farm’s dairy cattle to takeadvantage of the price premium from the cheese producers.In dairy breeds, an allele of the K-casein gene known as theB allele causes an increase in milk protein that has beenfound to help produce more of a better quality of cheese.This is why cheese producers will pay more forthe milk.

The farmer can only afford to buy one bull and two cowsfor the herd. The farmer locates three cows in separateherds that show a history of producing milk with higherthan normal protein content and two bulls whose daugh-ters produce high protein milk.

The farmer knows from experience that reading a pedigreeis difficult and many times can give incomplete informationabout the actual genotype of the cattle for the particulartrait of interest.

The farmer is ready to invest a great deal of money andwants to investigate the K-casein B gene allele in the cattleto be purchased. The farmer discovers that there is a newgenetic test that can determine if the K-casein B allele ispresent in cattle. The farmer asks that DNA samples fromthe five animals being considered for purchase be sent toyou for genetic analysis.

Your group is assigned to do the analysis and write a reporton the purchasing and breeding options for each of the fiveanimals. The samples are amplified using PolymeraseChain Reaction (PCR) technology and turned over to yourgroup for analysis.

Assume that the farm's current stock does not have the K-casein B allele and that the cattle being tested are unre-lated. A bull or cow with a genotype of AA is normal (no Ballele). AB has one B allele. A genotype of BB has two Balleles. The BB genotype will produce more milk proteinthan the AB genotype, and the AB genotype will producemore milk protein than AA. Depending on the breed ofcattle, there can be an increase of 3-12% in milk proteinproduction when the B allele is present.

You can download two simulations on how PCR works atwww.biotech.iastate.edu/publications/ed_resources/Laboratory_protocols.html.”

Student Instructions:These instructions are on the student handout titled Seefor Yourself: Agarose Gel Analysis of the K-Casein B Alleleon p. 133.

Step 1 (Optional)Put on medical gloves and wear them throughout theexperiment. The gloves will protect the DNAsamples from contaminants that may be onyour hands.

Step 2Your group has the five samples labeled M1(male 1),M2 (male 2), F1 (female 1), F2 (female 2), and F3(female 3). Keep the tubes upright throughout all thesteps of the experiment to keep the DNA off the sidesof the tube. Into each of the five tubes, pipette 3 μl ofthe restriction endonuclease Bgl l from the tubelabeled N. Use a fresh pipette tip when adding Bgl 1to each tube. To rinse the pipette tip and mix theDNA and Bgl 1, fill and unload by pushing andreleasing the pipette’s plunger three times. Label thefive tubes with a group letter or number that has beenassigned to your group and written in the upper righthand corner on the front of this instruction sheet.

Step 3Place the tubes in a rack provided by the instructorand incubate them at 37º C for 10-45 minutes asyour teacher directs. Bgl 1 is isolated from thebacteria Bacillus globigi. The restriction endonu-clease protects the bacteria from foreign DNA, suchas from a virus, by cutting it up and rendering itineffective. The endonuclease cuts the DNA (shownby arrows) at each site with the following sequences:

5'-GCCN NNN NGGC-3'3'-CGGN NNN NCCG-5'

N can be any nucleotide, but the location and orderof G (guanine) and C (cytosine) are very specific.



Step 4Remove the tubes from the incubator and keep themupright. If you are going to continue the lab the nexttime your class meets, place the tubes on a shelf inthe freezer until you are ready to complete the lab.You can remove them from the freezer at thebeginning of your next class period and thencontinue to step 5.

Step 5Into each of the five tubes, pipette 4 μl of bluemigration dye from the tube labeled D. Use a freshpipette tip when adding dye to each tube. To rinsethe pipette tip and mix the DNA and the dye, fill andunload by pushing and releasing the pipette’s plungerthree times. The blue dye is used to monitor themigration of the DNA during electrophoresis.

Step 6Go to the electrophoresis box and on a piece of paperrecord the identity and location of your samplesbefore loading them on the gel. The gel has lanes onwhich individual samples will be run, that corre-spond to the numbered lines on the sheet of paper.

Step 7Using a pipettor set for 20 μl, transfer your sampleinto the well of the appropriate lane in the gel. Placethe top of the pipette tip into the top of the well anddispense the 20 μl of solution into it slowly. Do notlet the pipette tip touch the bottom of the wellbecause it will puncture the gel. Discard the pipettetip in the designated container after a sample hasbeen put in the well and use a new tip for thenext sample.

The gel will be run and stained by your instructor.

Step 8After the gel is run, view the bands. From the DNApatterns on the gel, answer the Reflect and Applyquestions to determine how you would advisethe farmer about purchasing the cattle. Draw adiagram of your group’s results. Look at Figure 6 tosee how the gel results should appear.

Credit Notes

Atherly, Alan G.; Girton, Jack R.; and McDonald, John F.The Science of Genetics. Saunders College Publishing.1999

Basics of Marker Assisted Selection (BMAS). Julius vander Werf, Department of Animal Science, and BrianKinghorn, Twynam Chair of Animal Breeding Technolo-gies, University of New England.

Doggy DNA: The Power of PCR. 2000 Summer BiologyInstitute: Biodiversity. The Woodrow Wilson Founda-tion Leadership Program for Teachers.www.woodrow.org/teachers/bi/2000/Doggy_DNA/background_for_polymerase_chai.html

Kehrli, Marcus E. Bovine Leukoctye Adhesion Deficiency(BLAD) in Holstein Cattle. Virus and Prion Diseases ofLivestock Research Unit, National Animal DiseaseCenter-USDA-ARS, Ames, Iowa.

Marker-Assisted Selection: Applications to AnimalProduction. The Agbiotech Infosource. AG-WESTBIOTECH INC. Issue 39. October 1998

Olson, Tim. New Genes: Good and Bad. Departmentof Animal Sciences, University of Florida.

Polking, Gary F.. The Polymerase Chain Reaction:Introduction. Introduction to Molecular BiologyTechniques – Gen 542A. Iowa State University Officeof Biotechnology. Summer 2003

Suszkiw, Jan. Mapping the Way to Bovine Bounty. ARSNational Program Publication.

Van Eenennaam, Alison. Marker-assisted selectionbackgrounder. Agriculture and Natural ResourcesResearch and Extension Centers. University of Califor-nia-Davis, 2004 Science 75:3138-3142. 1997

PowerPoint® is a registered trademark of Microsoft Corporation.Knox® is a registered trademark of NBTY, Inc., used by Kraft Foodsunder license.

Reflect and Apply

1. After comparing the results of your gel to thepicture of ideal gel results on p. 133, explainhow each of the five cattle would affect thefarmer's goal to increase milk protein production.On the five worksheets M1, M2, F1, F2, andF3, construct a pedigree and use Punnett squares toshow how each of the five cattle would affect theprotein content in the milk when mated to thecurrent herd that does not have the B allele. Referto the pedigree and Punnett square diagrams youdo on the worksheets to show how your grouparrived at its conclusions.



M1The advantage of this bull (male) is that it carries oneB allele. See Figure 6. Figure 7 on the next pageillustrates a possible pedigree using bull M1 and thefarmer’s current stock.

Depending on the number of cows bred from thefarmer’s original herd, there would be a 50% chancefor each offspring produced to be heterozygous (AB).The farmer may recover some of the costs incurred bythe purchase by selling off the bulls and/or selling thesemen to other farmers through an AI program.

M2The M2 bull does not carry the B allele. It should notbe purchased. No pedigree and Punnett squarediagrams are needed.

F1This cow is homozygous for the B allele (BB).Any mating with an AA bull is going to produceoffspring that carry at least one B allele. By matingthe M1 bull with this cow, there is a 100% chance thatthe offspring would have at least one B allele and a50% chance that the offspring would be homozygousfor the two B alleles. See Figure 8.

F2This cow would have the same pedigree/Punnettsquare answer as the M1 bull.

F3This cow does not carry the B allele and should not bepurchased. No pedigree and Punnett square diagramsare needed.

Figure 6 – Ideal Gel Results

M1 M2 F1 F2 F3

2. Advise the farmer on the best breeding strategies.

There is only one choice of bull that could increasemilk protein, M1 (AB). The F1 cow is the best choicebecause all her offspring will have at least one Ballele. The F2 cow is the second choice because shecarries the B allele.



Figure 7

Figure 8

A B

A AA BA

A AA BA

A B

B BA BB

B BA BB

In Figures 7 and 8, the B allele that produces higher levels of the K-casein B protein is shadedblack. The male individuals (squares) and the female individuals (circles) that are completelyfilled with black will produce the highest levels of the protein.

131

Learning more about . . .

Iowa State University Extension and ISU Office of Biotechnology

Student Handout

The K-Casein B Allele in Cattle

Lesson Module II – Marker Assisted Selection MAS-4

Alleles of the K-casein protein have the greatest effect on thequality and quantity of cheese made from milk. Scott Bauer, ARS-USDA

Learn the Language

Alleles

Different forms of the same gene

Genotype

The genetic makeup of an individual, typicallyexpressed in alphabetical letters

_____________

… and justice for allThe U.S. Department of Agriculture (USDA) prohibits discrimination inall its programs and activities on the basis of race, color, national origin,gender, religion, age, disability, political beliefs, sexual orientation, andmarital or family status. (Not all prohibited bases apply to all programs.)Many materials can be made available in alternative formats for ADAclients. To file a complaint of discrimination, write USDA, Office of CivilRights, Room 326-W, Whitten Building, 14th and Independence Avenue,SW, Washington, DC 20250-9410 or call 202-720-5964.

Issued in furtherance of Cooperative Extension work, Acts of May 8 andJune 30, 1914 in cooperation with the U.S. Department of Agriculture.Stanley R. Johnson, director, Cooperative Extension Service, Iowa StateUniversity of Science and Technology, Ames, Iowa.

Introduction to K-Casein Proteins

A decade ago, milk produced by cows in the WesternWorld had a lower fat to protein ratio than consumerswanted. This excess milk protein was disposed of onworld markets in the form of dried skim milk. Today'strend in dairy production is to focus on more produc-tion of milk protein.

As pricing systems increasingly include payment for theamount of protein in the milk, breeding decisions havefocused on protein production. Two protein genes havebeen identified that affect milk protein production. It isnow relatively simple to determine the genotypes ofcows for these protein genes.

Studies conducted in the early 1970s indicated thatthe gene that controls the production of K-caseinhad the greatest effect on quality and quantity ofcheese production.

Two alleles of the K-casein gene have been identifiedas the A and B alleles. Cattle carry two copies of theA allele, denoted AA, or two copies of the B allele, BB,or one copy of each, AB. The B allele causes a substan-tial increase of milk protein yield in the cows carryingit. The B protein of K-casein results in a firmer cheeseand enhanced cheese yield from milk. The genotypeBB yields more protein than AB, which yields morethan AA.

Since the frequency of the K-casein B allele in dairybreeds is low (20%), it would be worthwhile to havebreeding programs that emphasize increasing thefrequency of the B allele in the dairy cattle population.Some countries have called for national breedingprograms for increasing the frequency of the B allele intheir dairy herds. Because of AI (artificial insemination)technology, a bull's genotype is of particular interest.

Iowa State University Extension and ISU Office of Biotechnology 133

See for yourself . . .

Student Handout

Which cattle should I buy?

A farmer in Iowa is interested in increasing the proteincontent in the milk of the farm’s dairy cattle to takeadvantage of the price premium from the cheeseproducers. In dairy breeds, an allele of the K-caseingene known as the B allele causes an increase in milk

protein thathas beenfound to helpproduce moreof a betterquality ofcheese. This iswhy cheeseproducers willpay more forthe milk.

The farmercan only

afford to buy one bull and two cows for the herd. Thefarmer locates three cows in separate herds that show ahistory of producing milk with higher than normalprotein content and two bulls whose daughters producehigh protein milk.

The farmer knows from experience that reading apedigree is difficult and many times can give incom-plete information about the actual genotype of the cattlefor the particular trait of interest.

The farmer is ready to invest a great deal of money andwants to investigate the K-casein B gene allele in thecattle to be purchased. The farmer discovers that thereis a new genetic test that can determine if the K-caseinB allele is present in cattle. The farmer asks that DNAsamples from the five animals being considered forpurchase be sent to you for genetic analysis.

Your group is assigned to do the analysis and write areport on the purchasing and breeding options for eachof the five animals. The samples are amplified usingPolymerase Chain Reaction (PCR) technology andturned over to your group for analysis.

Assume that the farm's current stock does not have theK-casein B allele and that the cattle being tested areunrelated. A bull or cow with a genotype of AA isnormal (no B allele). AB has one B allele. A genotypeof BB has two B alleles. The BB genotype will producemore milk protein than the AB genotype, and the ABgenotype will produce more milk protein than AA.Depending on the breed of cattle, there can be anincrease of 3-12% in milk protein production when theB allele is present.

You can download two simulations on how PCR worksat www.biotech.iastate.edu/publications/ed_resources/Laboratory_protocols.html.”

The picture below of an agarose gel shows the threepossible genotypes for the K-casein B allele.

Doing the Activity

Step 1 (Optional)Put on medical gloves and wear them throughout theexperiment. The gloves will protect the DNAsamples from contaminants that may be onyour hands.

Group Letter ___________________________________

Class/Section ___________________________________

Agarose Gel Analysis of the K-Casein B Allele


Keith Weller, ARS-USDA

AA AB BB

Ideal Gel of K-Casein B Genotypes

134

Student Handout


Step 2

Your group has the five samples labeled M1(male 1),M2 (male 2), F1 (female 1), F2 (female 2), and F3(female 3). Keep the tubes upright throughout all thesteps of the experiment to keep the DNA off the sidesof the tube. Into each of the five tubes, pipette 3 μl ofthe restriction endonuclease Bgl l from the tubelabeled N. Use a fresh pipette tip when adding Bgl 1to each tube. To rinse the pipette tip and mix theDNA and Bgl 1, fill and unload by pushing andreleasing the pipette’s plunger three times. Label thefive tubes with a group letter or number that has beenassigned to your group and written in the upper righthand corner on the front of this instruction sheet.

Step 3

Place the tubes in a rack provided by the instructorand incubate them at 37º C for 10-45 minutes asyour teacher directs. Bgl 1 is isolated from thebacteria Bacillus globigi. The restriction endonu-clease protects the bacteria from foreign DNA, suchas from a virus, by cutting it up and rendering itineffective. The endonuclease cuts the DNA (shownby arrows) at each site where the following sequencesoccur.

5'-GCCN NNN NGGC-3'3'-CGGN NNN NCCG-5'

N can be any nucleotide, but the location and orderof G (guanine) and C (cytosine) is very specific.

Step 4

Remove the tubes from the incubator and keep themupright. If you are going to continue the lab the nexttime your class meets, place the tubes on a shelf inthe freezer until you are ready to complete the lab.You can remove them from the freezer at thebeginning of your next class period and thencontinue to step 5.

Step 5

Into each of the five tubes, pipette 4 μl of bluemigration dye from the tube labeled D. Use a freshpipette tip when adding dye to each tube. To rinsethe pipette tip and mix the DNA and the dye, fill andunload by pushing and releasing the pipette’s plungerthree times. The blue dye is used to monitor themigration of the DNA during electrophoresis.

Step 6

Go to the electrophoresis box and on a piece of paperrecord the identity and location of your samplesbefore loading them on the gel. The gel has lanes on

which individual samples will be run, that corre-spond to the numbered lines on the sheet of paper.

Step 7

Using a pipettor set for 20 μl, transfer your sampleinto the well of the appropriate lane in the gel. Placethe top of the pipette tip into the top of the well anddispense the 20 μl of solution into it slowly. Do notlet the pipette tip touch the bottom of the wellbecause it will puncture the gel. Discard the pipettetip in the designated container after a sample hasbeen put in the well and use a new tip for thenext sample.

The gel will be run and stained by your instructor.

Step 8

After the gel is run, view the bands. From the DNApatterns on the gel, answer the Reflect and Applyquestions beginning on the next page to determinehow you would advise the farmer about purchasingthe cattle. Draw a diagram of your group’s results inthe space below. Look at the ideal gel on the previouspage to see how the gel results should appear.

Reflect and Apply

1. After comparing the results of your gel to the pictureof ideal gel results on p. 133, explain how each of thefive cattle would affect the farmer's goal to increasemilk protein production. On the five worksheets M1,M2, F1, F2, and F3, construct a pedigree and usePunnett squares to show how each of the five cattlewould affect the protein content in the milk whenmated to the current herd that does not have the Ballele. Refer to the pedigree and Punnett squarediagrams you do on the worksheets to showhow your group arrived at its conclusions.


135

Student Handout


Lesson Module II – Marker Assisted Selection

Credit Note

This activity was developed by Mike Zeller for theOffice of Biotechnology, Iowa State University, and isused with permission.

… and justice for allThe U.S. Department of Agriculture (USDA) prohibits discrimination inall its programs and activities on the basis of race, color, national origin,gender, religion, age, disability, political beliefs, sexual orientation, andmarital or family status. (Not all prohibited bases apply to all programs.)Many materials can be made available in alternative formats for ADAclients. To file a complaint of discrimination, write USDA, Office of CivilRights, Room 326-W, Whitten Building, 14th and Independence Avenue,SW, Washington, DC 20250-9410 or call 202-720-5964.

Issued in furtherance of Cooperative Extension work, Acts of May 8 andJune 30, 1914 in cooperation with the U.S. Department of Agriculture.Stanley R. Johnson, director, Cooperative Extension Service, Iowa StateUniversity of Science and Technology, Ames, Iowa.

MAS-5

2. Advise the farmer on the best breeding strategies.


Student Handout

Worksheet for M1 Bull

Name ________________________________________ Group ___________________________________


… and justice for allThe U.S. Department of Agriculture (USDA) prohibits discrimination in all its programs and activities on the basis of race, color, national origin, gender,religion, age, disability, political beliefs, sexual orientation, and marital or family status. (Not all prohibited bases apply to all programs.) Manymaterials can be made available in alternative formats for ADA clients. To file a complaint of discrimination, write USDA, Office of Civil Rights, Room326-W, Whitten Building, 14th and Independence Avenue, SW, Washington, DC 20250-9410 or call 202-720-5964.

Issued in furtherance of Cooperative Extension work, Acts of May 8 and June 30, 1914 in cooperation with the U.S. Department of Agriculture. StanleyR. Johnson, director, Cooperative Extension Service, Iowa State University of Science and Technology, Ames, Iowa.


Student Handout

Worksheet for M2 Bull

Name ________________________________________ Group ___________________________________





Student Handout

Worksheet for F1 Cow

Name ________________________________________ Group ___________________________________





Student HandoutLesson Module II – Marker Assisted Selection


Name ________________________________________ Group ___________________________________

MAS-9




Student Handout


Name ________________________________________ Group ___________________________________





Lesson Module II – Marker Assisted Selection Overhead Master: MAS-n

Genotype Key for

K-Casein in Cattle

AA = Homozygous AA allelesNo increase in milk protein

AB = Heterozygous AB allelesSome increase in milk protein

BB = Homozygous BB allelesMost increase in milk protein

AA AB BB


Lesson Module II – Marker Assisted Selection Overhead Master: MAS-o

Which Cattle Should I Buy?

M1 M2 F1 F2 F3

Ideal Gel

M1 = Bull

M2 = Bull

F1 = Cow

F2 = Cow

F3 = Cow


Lesson Module II – Marker Assisted Selection

A B

A AA BA

A AA BA

A B

B BA BB

B BA BB

The B allele that produces higher levels ofthe K-casein B protein is shaded black. Themale individuals (squares) and the femaleindividuals (circles) that are completelyfilled with black will produce the highestlevels of the protein.

K-Casein B Protein

Overhead Master: MAS-p

lesson module ii – marker assisted selection iilaboratory lesson plan: agarose gel analysis of the...

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