Biology 107 General Biology Labs 10 and 11 - University of Evansville

60the pipettor ARE sterile. This means that when youslowly insert the sterile tip into the growth medium toremove 0.99 ml of sterile broth and set up your dilutions,only the tip can touch the liquid or any part ofthe inside of the flask. If you accidentally “clunk” anypart of the micropipettor onto a sterile surface, such asthe inside of the flask containing the sterile broth, yourisk contaminating the broth for everyone else in yourlab. If you make this mistake, tell your instructor. Tominimize this potential problem, one member of eachgroup should carefully watch the persons using themicropipettor and visually help guide them as theyenter the flask.During this lab exercise, you will not refer toyour lab manual. Before class each week, eachstudent will individually prepare a protocol for thelab exercise. This protocol is a typed document thatlists the steps of your experiment, and explains whyeach step is important. Your protocol does not haveto be a long document, but it does have to be complete.Think back to the experiment in section B ofthe enzyme lab (page 39). A student protocol for thisexercise might read as follows.1. We will be carrying out four reactions, each withits own blank, so we will need eight test tubes foreach group. Blank tubes will contain potato extract,but no substrate (catechol) so that the color due to theextract can be subtracted from the total color in thereaction tubes to give the color due to product formation.Mark the tubes as indicated in the data table.2. Using glass pipettes to measure liquid volumes,make blank tubes containing water and potato extractaccording to the data table. Seal with parafilm andmix the blank tubes so that they are the same colorthroughout.3. Set the spectrophotometer to 540 nm. This wavelengthof light is absorbed by the product. The moreabsorbance we read, the more product is in the tube.4. Zero the spectrophotometer with Blank 1. Anyadditional absorbance in tube 1 is due to product formation.5. Begin making tube 1 by adding water and substrateto the tube. Do not add the extract (contains catecholase)until everything is ready.6. Start the reaction by adding potato extract, sealing,and mixing the tube. Note the time the reactionbegan. Allow the reaction to continue converting substrateinto product for three minutes.7. As the three-minute time approaches, mix the tubeagain, and read the absorbance at exactly three minutes.Record the absorbance and time in the datatable. The absorbance is in indication of how muchproduct was formed.8. Repeat steps 4 through 7 using tubes with differentamounts of potato extract. Be sure to use the correctblank with each reaction tube. Adding more enzymeshould increase the amount of product formed.This protocol, along with the data table, wouldallow you to do the experiment correctly. Writing itout beforehand would help you think about what youwere going to do and why. Your protocol is not completewithout an explanation of your experiment.You will need two protocols for week one -Counting Bacteria (including a dilution diagram),and Transformation. For the second lab week, youwill need a data table for transformation resultsas well as Plasmid Purification and Gel Electrophoresisprotocols. Remember, you will be usingyour written protocols, not your lab manual, so doa good job!Section A - Counting BacteriaHow many bacteria are in a given sample? Thisquestion arises in the clinical microbiology laboratory,in public health debates, and in many molecular biologyexperiments. How might you answer this question?The most convenient method is to spread thecells on an agar plate and then incubate the plate overnight.Wherever a cell lands on the plate, a bacterialcolony is present the next morning. For an example ofplating cells, see page 378 in your textbook.Competent cells have been prepared by yourinstructor, and are stored on ice in the tube labeled“Cells”. The cells must be kept ice cold at all times.

If they are allowed to warm even slightly, your experimentwill not work.Since your tube contains hundreds of millions ofcells, counting each and every one is not really possible.Instead, we will dilute the cells a known amount,count the cells in the diluted sample, and calculatethe number of cells in the original tube. The dilutioncalculations in the following example should becarefully reviewed before lab. If you want additionalexamples of dilution calculations, review your workfrom the first lab exercise.DILUTION SAMPLE CALCULATIONAssume that a tube has 1 ml of mediumcontaining 10 8 cells per ml. If you spreadall these cells on a plate, they would beimpossible to count. Since we can onlyreally count about 100 (10 2 ) cells on eachplate, we must dilute the cells in the originaltube one million (10 6 ) fold (10 8 cells inthe original tube divided by 10 6 = 10 2 cellson the plate) so that we can count them.Plating and counting the cells in a 10 6 folddilution of our original cells will allow usto multiply by one million to get back tothe number of cells per ml we started with.(100 cells on the plate multiplied by 10 6 =10 8 cells in the original tube)A one-million-fold dilution is impossible to carryout in a single step without using large quantities ofliquid. For example, diluting your original 0.1 ml onemillion fold in one step would require 99,999.9 mlor approximately 100 liters of medium. Instead, wewill use serial dilutions. Before beginning your dilution,you should watch the instructor demonstrate theuse of the micropipettors. Using these devices is animportant skill in the biology laboratory, and yourgrade depends on careful measurement.At the worktable, select three sterile microtubesfor each group and label them 10 -2 , 10-4, and 10 -6 .Working sterilely, use the micropipettor to place 0.99ml (990 µl) of sterile medium into each of the threesterile tubes. Remember that any bacteria introducedfrom your hands or elsewhere will throw off yourcount. Then, place 10 µl of your bacterial sample intoyour tube marked 10 -2 . Note that this is a 100 (10 2 )fold dilution of your original tube. Mix this tube well.Then, using a new sterile tip (why?) take 10 µl fromthis tube and place it in the tube marked 10 -4 , for a 10 4dilution. Mix, and place 10 µl from this tube into thetube marked 10 -6 for a 10 6 dilution.Now, still working sterilely, spread 200 µl of your10 -6 dilution onto an agar plate using the hockeystickmethod demonstrated by your instructor. Sinceyou want to assure that you can count the cells on theplate, make another counting plate with 20 µl of your10 -6 dilution.For your protocol, make a diagram explainingyour dilutions. Draw the tubes and plates you willuse, the amounts of liquid in each transfer, and howmany cells you expect in each tube or on each plate ifyour original solution has 10 9 cells per ml.Make sure the plates are properly labeled, andgive them to your instructor for overnight incubation.Arrange for your lab group to visit the lab tomorrowto count the bacterial colonies on the plate, and if necessaryto redo the experiment.Before you leave class, use your dilution diagramto think about how you will answer the followingquestion: what was the number of cells per ml in theoriginal tube? For example, if you count 17 cells in100 μl of your 10 -6 dilution, how many cells are ineach ml of this dilution? How many cells are in eachml of the 10 -4 dilution? How many cells per ml in theoriginal tube?IN YOUR LAB REPORT, STATE THENUMBER OF CELLS PER ml IN THEORIGINAL TUBE.Section B - Transformation61DNA has been prepared for you by your instructorand 1 µl of DNA solution will be placed into asterile 1.5 ml tube for each group. (One µl is 0.001ml and pronounced microliter. It is also referred to asλ or lambda.) Label the tube with your group numberand get ready to place 0.1 ml (100 µl) of cells fromthe ice water bath into the tube. It is essential that thecells stay ice cold and sterile at all times. Quicklyremove the tube labeled “Cells” from the ice bath,invert gently to mix, carefully open the cap and, using

the micropipettor, remove exactly 0.1 ml (100 µl)from the tube and place it in your labeled tube containingDNA. Gently mix the contents of the tube byflicking the side with your fingernail. Do not shake thetube or you will damage the bacterial cells.Immediately place the tube back into the ice bathand incubate it there for thirty minutes. This incubationallows the DNA to bind to the outside of the cell,and would be a good time to start your dilution experiment.After thirty minutes, heat shock the cells at exactly42°C for exactly ninety seconds, then return to icefor two minutes. This causes the cells to take upthe plasmid DNA. Then, add 0.9 ml of medium,and incubate at 37°C for a thirty to forty-five minuteoutgrowth. During the outgrowth, cells have anopportunity to express genes on the plasmid, makingthe protein responsible for conferring ampicillin resistance.Plate 0.1 ml of your transformation mix ON APLATE CONTAINING AMPICILLIN using the samemethod you used in your dilution. Plate 0.01 ml onanother plate, in case your first plate has too manycolonies to count. Count the transformed coloniestomorrow.IN YOUR LAB REPORT, STATE: 1)THE NUMBER OF CELLS IN YOURTRANSFORMATION TUBE; 2) THENUMBER THAT TOOK UP THEPLASMID; AND 3) THE TRANSFOR-MATION EFFICIENCY (EXPRESSEDAS A DECIMAL).Section C - Counting ColoniesWhen you return to class to count your cells,remember that wherever a single cell landed on thesurface of the plate, a colony of cells will be presentthe next day. When you count these colonies, you arecounting all of the cells that were able to live on theplate where they were spread. You are estimating twoquantities: the number of cells in the “Cells” tube, andthe number of cells that took up the plasmid. For eachof these estimates, you have two plates. You shouldsee a 10-fold difference between similar plates.Example: If you count 37 colonies on the ampicillinplate which received 10 µl of transformed cells,you would expect about 370 colonies on the platewhich received 100 µl. If, however, you counted 11colonies on the plate which received 100 µl of transformedcells, you may find no colonies on the platewith the smaller volume. You expect a 10-fold difference,but you won’t always find it. In this lab, youshould count all of the colonies that are possible tocount Enter the number of colonies you count in thespace below. If there are so many colonies on a platethat they are too close together to distinguish, make anote of that on your data sheet and use the count fromthe companion plate for your calculations.After you count the colonies on your plates, besure to WASH YOUR HANDS WITH SOAP beforeleaving lab. Save your plates for next week’s labexercise (See Section D).PlateCells, 10 6 dilution200µlCells, 10 6 dilution20µlTransformation100µlTransformation10µlNumber of ColoniesIn order to know how well your experimentworked, you will need to compare your results to theresults of others. Design a table to collect the transformationand cell count results from all of the groupsin your lab section. Bring it to class next week.Section D - Plasmid Purification62When biologists introduce a plasmid into E. coli, itis usually because they want to use the bacterium asa means of storing and amplifying the plasmid. If theplasmid carries a DNA insert, then the insert is amplifiedalong with the rest of the plasmid. This is a goodway to make a large number of copies of an insert forfurther analysis. It is only useful, though, if there is a

63convenient way to recover the plasmid from the bacteria.Remember, in the transformation experiment,you introduced plasmid molecules into bacterial cells.Each cell that took up a plasmid became a colony containingmillions of cells after overnight growth. Sinceeach of these cells has between ten and one hundredplasmid molecules, large amounts of plasmid (andinsert) can be prepared quickly.During the second week of this lab exercise youwill grow bacterial cells containing your plasmid,purify the plasmid DNA and visualize it using anagarose gel. Plasmid purification involves a numberof steps, as described below, but the principle is verysimple. Cells are collected, broken open, and plasmidDNA is separated from all of the other moleculesin the cell. This procedure is outlined in Figure 3.Figure 4 on the following page shows an illustratedversion of the steps outlined in Figure 3.The day before your lab period, someone from yourgroup should come to the laboratory at the scheduledtime. Make sure you have prepared for this meeting- you should know all the details about what you aredoing, and why. Your instructor will have prepareda number of culture tubes containing sterile liquidmedium that contains ampicillin. Working sterilely,inoculate a culture tube with a single bacterial colony.Simply touch the inoculating loop first to the colonythen to the liquid in the tube. It is not necessary tomove the whole colony, or even a visible clump ofcells. Place the tube in the shaking water bath asdirected, and allow to shake overnight at 37° C.At the beginning of your lab period, each groupshould take one 1.5 ml microtube, label it with theirgroup number, and use a pipette to transfer 1.5 ml ofthe bacterial culture into the tube. Then, place thetube in the microcentrifuge, and your instructor willspin the tubes so that the cells form a pellet on thebottom of the tube. Each group should then recovertheir tube, pour off the medium without disturbing thepellet, and add another 1.5 ml of the same bacterialculture to the tube containing the pellet. After anothercentrifugation and complete removal of the medium(also called the supernatant) the tube will contain onlythe cells from 3 ml of bacterial culture.Next, we will add 250 µl of cell resuspension solutionto the pellet. Use the vortex machine to getall of the cells back into suspension. When you areready, add 250 µl of cell lysis solution and mix byinverting the tube four to five times. This solution hasa very high pH and contains sodium dodecyl sulfate, astrongly amphipathic detergent that dissolved the cellmembrane, releasing the contents. When you add thisreagent, you should see the solution clear somewhatand become very viscous. The viscosity is caused bythe bacterial chromosome, which we will precipitateand discard shortly. Treat the contents of the tubegently after lysis - DO NOT VORTEX. After yourlysate has cleared, add 10 µl of alkaline protease solutionto digest cellular protein, and incubate for fiveminutes at room temperature.Figure 3. Outline of Plasmid Purification.Add 350µl of neutralization solution to the lysate,and mix gently (do not vortex). This lowers the pHand most lipids precipitate (come out of solution).

64Since the bacterial chromosome is attached to themembrane, it also precipitates in this step. Yourinstructor will centrifuge the tube for ten minutes,and this will again result in a pellet and a supernatant.This time it is the supernatant that contains theplasmid DNA. Move to the vacuum manifold (Figure4 on the next page), obtain and label a minicolumnwith your group number, install the minicolumn ontothe vacuum manifold as directed, and carefully pouryour cleared lysate into the column. The column containsresin that binds plasmid DNA. You will attachyour plasmid DNA to the column and then wash awayother impurities.Apply vacuum using the stopcock on the vacuumstem so that your lysate is drawn through the column.When all the liquid has been drawn through, release thevacuum. Then add 750 µl of column wash solution,apply vacuum to draw it over the column, and releasethe vacuum. Repeat the wash step, using only 250 µlof column wash solution for the second wash. Whenall of the wash solution has been removed from thecolumn, release the vacuum, and transfer the columnto a spin collection tube. Your instructor will spin thecolumn briefly in the microcentrifuge to remove anyremaining liquid. Then, transfer the column to a new1.5 ml microtube (labeled with your group number),add 100 µl of water to the column, and allow yourinstructor to centrifuge the water through the column.Since the DNA does not bind to the column resin inthe presence of pure water, it is released, and travelswith the water into the centrifuge tube. You may nowdiscard the column.Figure 4. Wizard SV Illustrated Protocol. Reproduced with permission from Promega Notes 59:10 (1996).

65Section E - Gel ElectrophoresisCAUTION: UV light and ethidium bromide areboth hazardous. Listen carefully when your instructorexplains how to minimize your exposure.A glance at the tube with your plasmid shouldconvince you that a small volume of water containingplasmid DNA is not easy to distinguish from any othersmall volume of water. We need a way to visualizethe DNA. To determine whether you have correctlypurified your plasmid, combine 10 µl of your plasmidsolution with 5 µl of blue loading buffer in a new500µl microtube. Be careful, this buffer will stainyour skin and clothing. Mix gently, centrifuge for 5seconds, and load all 15 µl into a well of the agarosegel your instructor has prepared. Once all groups haveloaded their DNA on the gel, an electric field will beapplied, and negatively charged DNA will movetoward the positive electrode. The gel matrix retardsthis movement, with larger DNA molecules retardedmore than small molecules. The result is separationof DNA molecules by size (Figure 5). Your instructorwill include on the gel a sample of the correct plasmid,and yours should run at the same size.DNA will be visualized using a UV transilluminator.The gel matrix prepared by your instructor containeda compound called ethidium bromide. Thismolecule binds strongly to DNA, and the bound complexfluoresces orange in the presence of UV light. Wewill place the gel on a UV light source, and any DNAin the gel will begin to glow brightly. Your instructorwill post a photo of your gel on the course web site foryou to include in your lab report.Figure 5. DNA separated by size on an agarosegel. This gel contains 20 lanes. Each sample isloaded into a well near the top of the gel. An electricfield is applied, and the molecules move downwardin each lane, with small molecules movingfastest. DNA is visualized under UV light in thepresence of ethidium bromide. The far right andleft lanes contain standards of known size.Lab AssignmentThis assignment is to be handed in the week of November 28, at the beginning of lab (beforeyour quiz). Each student must complete this assignment individually.Write a brief introduction and a methods section explaining what you did in this experiment. Make surethese sections cover transformation, selection, plasmid purification and gel electrophoresis. Then, write a resultssection in which you report the number of cells per ml in the original tube of competent cells (the tube labeled“Cells”). Use this number to determine how many cells you used for the transformation. Calculate how many ofthese cells were transformed. Why must you use data from other lab groups in your section to get an idea of howwell your experiment worked? Your lab report will not be complete without an analysis of your data compared toclass results. Return to section D of the enzyme lab exercise (pages 45-46). How can you use the class mean andstandard deviation to tell whether your results make sense? Include in your results section a printed photo of youragarose gel showing that you recovered the same plasmid you received from the instructor in the first lab period.All text material for this assignment must be submitted to www.turnitin.com.