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

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$Math 566 WI 2011 Midterm 1 Review and Practice Problems ...$

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
Page 3: 60the pipettor ARE sterile. This me
Page 7 and 8: 64Since the bacterial chromosome is

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

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