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An ImportantLaboratory Skill for MolecularBiology Downloaded from http://online.ucpress.edu/abt/article-pdf/66/4/291/51841/4451672.pdf by guest on 01 October 2021

JON S. MILLER MICHELLE. SASS SUSAN J. WONG JAMES NIENHUIS

A \ Imosttwo decadesago, few peoplewere nology is becoming a familiarword and has a signif- aware of the term biotechnology.In fact, physicians icant influence on many of the decisions people and pharmacistshad to receive additional instruc- make regardingtheir personallives. tion to appreciatethe new technologiesbeing devel- oped and utilized by researchlaboratories (Steggles, One areaof biotechnologythat is gainingpopu- 1987; personal communication with pharmacists larityin -basedscience classes is molecu- and physicians). For the most part, the teaching of lar (Zeller,1994). Molecularbiology is a dis- biotechnology took place in upper-levelundergrad- cipline that combines theories of physical science uate and graduatecourses, and the laboratoriesof with biology in an effort to understand the basic colleges and universities. Today, biotechnology is mechanisms of living organisms. DNA is the pri- discussed in elementary schools, middle schools, mary biological molecule in molecular biology and high schools as well as communitycolleges, jun- investigations.The techniques used in the study of ior colleges, and universities.It is clear that biotech- DNA have become an integral part of nearly every majorfield of biological science. Sensationalmedia JON S. MILLER is in the Departmentof BiologicalSciences at coverageof high profile trialsand the advent of tele- Northern Illinois University, DeKalb, IL 60115; e-mail: vision shows featuringforensic sciences have con- [email protected] E. SASSis in the Departmentof tributedto a tremendousinterest in the application Horticulture at the University of Wisconsin, Madison, WI of DNA technology. More importantly, however, 53706. SUSANJ.WONG is in the ScienceDepartment at DeKalb with recent advances in the Human and Plant in the High School, DeKalb, IL 60115. JAMESNIENHUIS is Genome Projects, DNA fingerprinting (forensic Department of Horticultureat the University of Wisconsin, Madison. science), genetic engineering, and cloning, an

MICROPIPETTING291 understandingof DNA technologyis becoming increas- the emergenceof affordablelaboratory equipment, biol- ingly important to everyone. Recognizing this strong ogy teachers can now instruct their students with a interest,biology teachershave made effortsto integrate hands-onapproach in the laboratoryinstead of a lecture DNA science and technology into the biology curricu- session or a "paperand pencil"activity. However, to pro- lum. Manybiology teachersrecognize the importanceof vide appropriatelaboratory experiences, emphasis must providingstudents with an understandingof the basic be placed on developing the skills requiredto success- principles and applications of molecular biology fully carryout the hands-onlaboratory activities associ- through an inquiry-based curricula. Several teacher- ated with molecularbiology. workshops in biology have focused on the training Pipettingis an essential skill students need to mas- introduction of molecular biology techniques and the ter to successfullyexecute laboratoryactivities in many theories underlying them (Cullis, 1998; Bloom, 2001; fields of science, including molecular biology (Figure Hoff, 2002; Sabir, 2002). With the development of 1). Molecularbiology laboratoriesutilize micropipetting to in opportunitiesfor teachers participate trainingpro- instruments (micropipettors) as tools to accurately grams, coupled with reliable laboratoryactivities and transfervery small volumes (microliters)of liquid from one containerto another.Skillful use of a micropipettor is a criticalcomponent of molecularbiology laboratory

procedures. To perform this skill effectively requires Downloaded from http://online.ucpress.edu/abt/article-pdf/66/4/291/51841/4451672.pdf by guest on 01 October 2021 practice. Often, failure to perform an accurate micropipettingstep in the laboratory procedure will 4 ~ ~~~4 result in poor results or no results at all. Yet, there are few resourcesbiology teachers can access that explain the theory and practice of micropipetting.Companies that marketmicropipettors include instructionbooklets on the use and care of their product. The instruction booklet provided by Rainin (Rainin Instrument Co., Inc., Oakland,CA) serves as an example, but does not provide practice exercises for developing the skill in using a micropipettor.Therefore, we recommend that everyone interested in incorporating micropipetting into their science curriculumfirst read the manufactur- Figure1. er's instruction booklet for technical information Photographof studentspracticing their pipetting technique regardingthe micropipetterand its operation,and then witha micropipettor.(Photograph by J.S. Miller) providestudents with practiceactivities that allow them to develop their skills in using a micropipettor. In this paper, we describe the use and care of a micropipettor Table1. (Table 1) and present activities Maintenanceand careof the micropipettor:Important things to designed to help biology students rememberregarding the use and careof the micropipettor. conceptualize working with small volumes and develop their skill in 1. Applyuniform pressure to the plunger. using this importanttool for molecu- 2. Releasethe plungerslowly and with smoothness. lar biology. 3. Donot immerse the pipettetip completely into a solution. Micropipetting 4. Maintaina minimal angle when pipetting. Technique 5. Neverlay the micropipettoron itsside when the pipettetip contains liquid. 6. Toobtain the mostaccurate measured volume, use the micropipettorbest suited for Becoming Familiar with yourpipetting needs. the Micropipettor 7. Avoiddropping and rough handling of the micropipettor. Before students begin working 8. Ifliquid has gone up into the barrelof the micropipettor,inform one of the instructors. with a micropipettor, they should 9. Seethe manufacturer'sinstruction booklet for more specific information regarding the take the time to become familiar useand care of the micropipettor. with the basic features (Figure 2A and B) and how they function.

292 THEAMERICAN BIOLOGY TEACHER, VOLUME 66, NO.4,APRIL 2004 Micropipettorscan dispense various volumes. Generally, Plunger Button button indicates the plunger Tip ejecter the maximum volume A. w / (microliters)that the pipette l l Volume 1 1 ~~~button is designed to handle. For adjustment example, the Gilson P20 \ AI Pipetman P-20 (Rainin 1 3s \ 4 < Instrument Co., Inc., _ 10s ~~~Digital q , , j ~~volume ___l Oakland, CA) will handle 3 1 s indicator up to 20 microliters. The volume indicator is read 4 Ol1s from top to bottom (Figure 2B). For P-2, P-10, P-20, P- 13.4 ulStils 100, and P-200, black digits indicate microliters and red ejector arm digits indicate tenths and Downloaded from http://online.ucpress.edu/abt/article-pdf/66/4/291/51841/4451672.pdf by guest on 01 October 2021 hundredths of microliters. For P-1000, red digits indi- cate milliliters and black digits indicate microliters. B. Tip ejecter Not all micropipettors have P200 P1000 colored digits on their vol- ume indicators. 0 loos 0 iooos 7 lO s 2 lOOs Student Activities 3 is 8 lOs Once the students have become familiar with the 73 ul 280 ul parts and functions of the Polypropylene micropipettor,they can move disposable tip on to the activitiesdescribed below to further develop their micropipettingskills. Figure2. A.General diagram of a typicalmicropipettor. Micropipettors come in various sizes ranging from 5 Materials plcapacity to 5000pl capacity. Generally, 20 p1,200 pl, and 1000 pi size are most commonly used in molecularbiology laboratory activities. B. The volume indicator is readfrom top to bottom.For 20 * Small beakers (10 to plcapacity, and 200 pl capacity micropipettors, black digits indicate microliters and red digits indi- 50 ml capacity) catetenths and hundredths of microliters.For 1000 p1l capacity micropipettors, reddigits indicate * Wax paper (10 x 10 millilitersand black digits indicate microliters. Not all micropipettors have colored digits on their cm squares) volumeindicators. (Diagram by B. Ball) * Micropipettors P-20 (size note: the activi- ties can be modified * Petridish (100 x 15 mm) to accommodatelarger pipettors i.e. P-100,P-200, P-1000.) * Glycerol * Pipettor tips (appropriate for the type of * Food coloring (blue, red, yellow) micropipetterbeing used) * 0.5 ml polypropylenemicro-centrifuge tubes Conceptualization of Small * 1.5 ml polypropylenemicro-centrifuge tubes Volumes * Agarose(or agar) The exercisepresented here is designed to help stu- dents conceptualizeworking with microlitervolumes. * TBE(Tris-Boric Acid-EDTA) for agaroseprep only Studentsshould be familiarwith a two-literplastic bev- * Gel comb eragecontainer and thus be able to visualize a one-liter

MICROPIPETTING293 plastic beverage or a one-quart milk carton (which approximatesa liter). Challenge the students with the followingquestions. The answersare provided later in the Solutions section. 1. How many microlitersdoes it take to make a liter? 2. If you could move 1 microliterof water per sec- ond, how many seconds would it take to fill a 1- liter bottle with water? 3. How many days is this equivalentto?

Basic Use of the Micropipettor In this exercise,students will become familiarwith the operationof a micropipettor.Use an adjustablevol- Downloaded from http://online.ucpress.edu/abt/article-pdf/66/4/291/51841/4451672.pdf by guest on 01 October 2021 ume micropipettorthat has a capacityfor 20 pt volumes. Also, have a small amount (10 ml) of water availablein a beaker and an empty beaker or small to receivethe transferof liquid. Figure3. 1. Hold the micropipettorin one hand (Figure 3). Photographofstudent adjusting the volume of a micropipettor. With the other hand, turn the volume adjust- (PhotographbyJ.S. Miller) ment knob to the desired setting (Figures 2 A and B).

2. Attach a new disposable tip to the pipette shaft. Practice Pipetting Small Volumes Be sure the tip is properly attached and has a good seal. The purpose of this exercise is to further develop skills using a micropipettorand to gain appreciationfor 3. Press the plunger to the first stop (Figure 4B) workingwith small volumes of liquid. whereyou feel a slight resistance.This represents the volume displayed on the digital indicator Procedure (Figure2B). 1. Pipetteand then dispense 10.0 1tland 5.0 [il sam- 4. Holding the micropipettorvertically, immerse ples of water on a piece of wax paper (10 x 10 the tip a few millimetersinto the sample while cm). Adjust the volume of the micropipettorto holding the plungerat the first stop. 15.0 >1.. 2. Depress the plunger to the first stop. While 5. Allow the plunger to slowly return to the UP releasingthe plungervery slowly, draw up both position (Figure 4A). Remember;do not let it the 10.0 p1volume and the 5.0 p1volume within "snap"to the up position. Then carefullywith- one release (stroke) of the plunger.Keep practic- draw the tip from the sample making sure there ing until you can do this without leaving any liq- are no air bubbles. uid behind on the wax paper.If air is allowed to 6. To dispense the liquid,gently touch the tip to the be introducedinto the micropipettorafter the 10 side of the receiving vessel. Press the plunger tl volume, students will not be able to draw up past the firststop to the second stop (Figure4C). all the remainingliquid. With the plungerfully pressed, withdrawthe tip 3. Repeat the above exercise using decimal vol- carefully,wiping residualdrops againstthe vessel umes; e.g., 6.2 p1 and 10.8 R1. Adjust the wall. micropipetterto 17.0 p1. 7. Allow the plunger to slowly return to the UP Transferring Small Volumes position. In this exercise, students will gain experience in 8. Discardthe tip by depressingthe tip ejectorbutton. performing a technique known as "gel loading."

294 THEAMERICAN BIOLOGY TEACHER, VOLUME 66,NO.4, APRIL 2004 develop skill in using the micropipettorfor "loading" wells in an agarosegel (or agar)similar to those used for electrophoresis.

Procedure Students will dispense a solution of glycerol and food coloring from a 0.5 ml test tube to a well in the practicegel. 1. Load8 [tl of red dye into the firstwell of the prac- tice gel. 2. Load 10 tl of blue dye into the second well of the practicegel. B. 3. Load 15 tl of yellow dye into the thirdwell of the practicegel. 4. In a 0.5 ml test tube, pipette 5 RIof blue dye and Downloaded from http://online.ucpress.edu/abt/article-pdf/66/4/291/51841/4451672.pdf by guest on 01 October 2021 5 p1 of red dye. Then load 10 p1 into the fourth well of the practicegel. 5. In a 0.5 ml test tube, pipette 2 p1of blue dye and 6 p1 of yellow dye. Then load 8 R1into the fifth well of the practicegel. 6. In a 0.5 ml test tube, pipette 4.2 pt red dye and 15.8 pt yellow dye. Then load 20 p1into the sixth C. well of the practicegel. The finishedproduct should look similarto Figure5.

Solutions: Activity A 1. How many microliters does it take to make a liter?Answer: 1,000,000 ,ul. 2. If you could move 1 microliterof water per sec- ond, how many seconds would it take you to fill a 1-liter bottle with water? Answer: 1,000,000 seconds. Figure4. 3. How many days is this equivalent to? Answer: A.The photograph shows the up position of theplunger button. 1 liter x 1000 milliliters/liter x 1000 micro- Thisrepresents the starting position for proper pipetting tech- liters/milliliter x 1 second/microliter x 1 nique.B. The photograph shows the plunger button depressed minute/60 seconds x 1 hour/60 minutes x 1 tothe 1st stop position. While holding the plunger button in this day/24 hours = Time in days to fill the liter. Therefore,((1000 x 1000)/(60 x 60 x 24)) days positionand inserting the pipettor tip into the sample, then = (1,000,000/86400) days = 11.57 days. slowlyreleasing the plunger button to the up position, the desiredvolume of sample will be drawn up into the pipettor tip. C.The photograph shows the plunger button depressed tothe Teacher Preparation 2ndstop position. Depressing the plunger button beyond the 1st stopposition to the2nd stop position will result in expelling any Practice Loading Dye Preparation remainingliquid from the pipettor tip. (Photographs byB. Ball) Preparethree 6 ml S0% glycerolstocks (3 ml glycerol, 3 ml distilledH20). Add 4 drops of food coloring(blue, red, and yellow). Mix well. Aliquot 200 p1 into 0.5 ml Studentswill practicedrawing up microlitervolumes of tubes. This will be enough for 30 tubes per color.These liquid and then dispense them in the well of a practice can be reused.For short-termstorage, place in the refrig- gel. The objectiveis for students to gain familiarityand eratorand for long term storage,place in the freezer.

MICROPIPETTING295 Practice Gel Plate Preparation Conclusion To make practicegel plates, agarose,IX TBE(Tris- The micropipettoris an important tool in many BoricAcid-EDTA) buffer, 100 x 15 mm petri dishes, and fields of science.As a startingpoint for the introduction mini electrophoresisgel combs are needed. To prepare of molecular biology, developing laboratory skills 500 ml 1%agarose, heat 500 ml IX TBEbuffer and 5 g becomes an importantconsideration. The natureof the micropipettormakes it imperativefor one to know how to use the instrument without invalidatingits accuracyand precision. In general, it is difficult to assess whether or not poor or unreliableresults are due to operator error or the instrument itself. Therefore,it is worthwhile taking time to allow stu- dents to practice and develop familiaritywith a micropipettor before they engage in the actual molecularbiology laboratoryactivities.

Acknowledgments Downloaded from http://online.ucpress.edu/abt/article-pdf/66/4/291/51841/4451672.pdf by guest on 01 October 2021 The authors wish to thank Barb Ball, Department of Biological Sciences Graphics Laboratory,Northern Illinois University, for her technical assistance with the graphics; and Federico Luis Iniguez Luy, Department of Horticulture,University of Wisconsin-Madison,for Figure5. his technical assistance in developing the proce- Photographof studentloading a wellof a practicegel. dures described in this paper. In addition, we wish to express our L (Photographby J.S. Miller) appreciationto the National Science Foundation (NSF Grant # 98-19051) for support- ing Michell Sass. This work was supported in part by the Department of Horticulture, University of Wisconsin-Madison; the Department of agarosein a 1 L beaker or flask on a (or in a Biological Sciences, Northern Illinois and the DeKalb microwaveoven) and bring to a rapid boil. Stir the 1% University; High School Science Department, DeKalb Public agarose solution occasionally to ensure uniform melt- School District #428. ing. Caution should be taken when stirring,as the 1% agarosesolution may contain trappedpockets of steam and can cause severe burns. Cool 1% agarosesolution to 60 C and pour into petridishes until properdepth of References gel comb is met (approximately3 mm or half the depth Bloom, M. (2001). Molecular of the gel comb). The depth can differwith various gel biology and technology. The AmericanBiology Teacher, 63(8), 557-560. comb manufacturers.(Excess agarose solution can be set into a 60 C waterbath to keep it in liquid state or it Cullis, C.A. (1998). A biotechnology experience resource can be stored in a solid state in the refrigerator.) center in Northeast Ohio. TheAmerican Biology Teacher, 60(3), 182-184. Waitapproximately 25-30 minutesbefore removing the gel combs from the petri dishes. Prematureremoval Hoff, D. (2002). Biology classes analyzing genetics. of the gel combs can cause the wells in the agaroseto EducationWeek, March 6, pp 19-21. collapse. Five hundred ml 1% agaroseyields approxi- Sabir,A. (2002). High school teachers and students are put mately 10 plates. These can be rinsed and reused one to work. Utah State University Biotechnology, 7(1), 1-3. time. Similarplates can be made using agar(prepared as the packagedirects). However, these do not rinse effec- Steggles, A.W. (1987). Teaching biotechnology to medical tively and cannot be reused. Severalbiological supply students: Is there an easy way. The Ohio Journal of companiesoffer consumables and equipmentfor molec- Science,87(5), 158-161. ular biology in their catalogs. Carolina Biological Zeller,M.F. (1994). Biotechnologyin the high school biolo- (CarolinaBiological Supply Co., Burlington,NC) is one gy curriculum:The future is here! TheAmerican Biology companythat offersa kit for practicingmicropipetting. Teacher,56(8), 460-464.

296 THEAMERICAN BIOLOGY TEACHER,VOLUME 66,NO. 4, APRIL 2004