Lab 10: Bacterial Transformation, part 2, DNA plasmid preps, Determining DNA Concentration and Purity

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1 Lab 10: Bacterial Transformation, part 2, DNA plasmid preps, Determining DNA Concentration and Purity Today you analyze the results of your bacterial transformation from last week and determine the efficiency of your transformation. You will also perform a small-scale DNA plasmid preparation, which involves isolating a DNA plasmid from bacterial cells (effectively the opposite of transformation). Finally, you will use the spectrophotometer to determine the concentration and purity of a DNA sample. Activity 10a Transformation, Part 2: Calculating Transformation Efficiency Purpose The purpose of this activity is to observe the results from the bacterial transformation you performed last week. You will observe the four agar plates to see if the results match the predictions you made last week. You will also calculate the efficiency of your transformation to quantitatively determine the relative success of the transformation procedure. Procedure 1. Use a data table similar to Table 10.1 below to record data from your bacterial transformation experiment. Observe your group s plates using the UV lamp and record what you see on each of the four plates. In your observations, note whether you see bacterial colonies, and whether the colonies fluoresce (glow) green under the UV light. Table 10.1 Bacterial Transformation Observations Plates +pglo, LB/amp Observations +pglo, LB/amp/ara -pglo, LB/amp -pglo, LB 89

2 2. Calculate your transformation efficiency. There are several steps involved in calculating transformation efficiency. Step 1: Determine the total number of green fluorescent cells. Place your LB/amp/ara plate near the UV light source. Each colony on the plate can be assumed to be derived from a single cell. That reproduced many times to form a colony. Therefore, counting the number of green fluorescent colonies tells you how many cells were transformed. Step 2: Determine the total amount of pglo plasmid DNA added to the bacterial cells on the LB/amp/ara plate. a) In our transformation, we used 10 µl of pglo DNA solution (approximate amount in the loopful in step 5) at a concentration of 0.08 µg/µl. Calculate the total µg of DNA that we used in the experiment. Total amt. of DNA (µg) = (concentration of DNA (µg/µl) x volume of DNA in µl) = b) Determine the fraction of this total amount of DNA that was actually spread onto the LB/amp/ara plate. On your LB/amp/ara plate, you spread 100 µl of (+pglo) transformation suspension from a total volume of ~510 µl. Fraction of DNA spread on plate = Volume spread on LB/amp/ara plate Total volume in transformation tube = c) Determine the number of micrograms (µg) you spread on the LB/amp/ara plate. To do this, multiply the total amount of DNA used in the experiment (from part a above) by the fraction of DNA you spread on the LB/amp/ara plate (from part b above). pglo DNA spread (µg) = Total amt of DNA used (µg) x fraction of DNA on plate = d) Calculate transformation efficiency. Total # of cells transformed (colonies counted) = Transformation = total # of cells growing on LB/amp/ara plate = Efficiency amount of DNA spread on LB/amp/ara plate = transformants/µg DNA 90

3 Activity 10b DNA plasmid prep ( Mini-prep ) Purpose In this activity, you will perform a small-scale DNA plasmid preparation, called a miniprep. You will be isolating the pglo plasmid back out of E. coli cell cultures essentially, the opposite of the bacterial transformation that you performed last week. Background DNA preps are preparations of DNA, frequently plasmid DNA, from transformed cells. Depending on the scale of the prep (how much bacterial liquid culture is grown, how much plasmid DNA yield is expected, etc.), the preps are called miniprep, midiprep, maxiprep, megaprep, or even gigaprep. DNA plasmid preps are an important way of generating DNA plasmid stocks for long-term storage to be used in future transformations and other techniques. Once bacterial cells have been transformed, they are grown under selection (i.e. in the presence of antibiotic) so that each cell must carry the plasmid to survive. Then, as the cells replicate in culture, the DNA plasmid is replicated as well, generating lots of copies of the DNA plasmid. The plasmid preparation then purifies the plasmid DNA from the bacterial cells. Resuspend Lyse neutralize Procedure 1. Pellet cells. Spin bacterial cultures at high speed in a microcentrifuge for about 5 seconds to pellet the bacterial cells. 2. Pour the supernatant into a waste beaker containing bleach. Remove any residual LB nutrient broth using a micropipet and put in waste beaker along with the micropipet tip. 3. Resuspend cells. Add 250 µl of Buffer P1 and resuspend cells until there are no cell clumps visible. 4. Lyse cells. Add 250 µl of Buffer P2 and gently invert the tube 4-6 times. The solution should become viscous and partly clear. Do not allow the lysis reaction to proceed for more than 5 minutes. (Buffer P2 contains a mixture of strong base (NaOH) and detergent (SDS) to lyse the bacterial cells and denature proteins). 5. Neutralize lysis. Add 350 µl of Buffer N3 and invert the tube immediately but gently 4-6 times the solution should become cloudy. (Buffer N3 contains acid to neutralize the basic lysis solution from the previous step, as well as a high salt concentration that will cause the chromosomal DNA, denatured proteins, and cellular debris to precipitate). 91

4 6. Centrifuge for 10 minutes at high speed. During the centrifugation, place a spin column in a 2 ml collection tube. 7. Bind. Apply the supernatant from step 6 to the spin column by micropipetting. In this step, the plasmid DNA binds to the column; other molecules should not bind and flow through. 8. Centrifuge seconds. Discard the flow-through. 9. Wash column by adding 500 µl of Buffer PB. Centrifuge seconds. Discard the flowthrough. 10. Wash column again with 750 µl of Buffer PE. Centrifuge seconds. Discard the flowthrough. 11. Centrifuge an additional 1 minute to remove residual wash buffer. 12. Elute. In this step, the DNA is released from the column by adding a low-salt or no-salt liquid. Place spin column into a clean 1.5 ml microcentrifuge tube. Add 50 µl of Buffer EB (10 mm Tris, ph 8) to the center of the column. Let stand for 1 minute, and centrifuge for 1 minute. 92

5 Activity 10c Determining DNA Concentration and Purity Purpose In this activity, you will learn how to calculate the concentration of a DNA sample using the spectrophotometer. You will also use the spectrophotometer to determine the degree of purity of a DNA sample. Background Whenever technicians purify DNA samples, they then need to determine: 1) how much DNA they obtained, and 2) how pure the DNA is (meaning, are there contaminating molecules, usually protein or RNA, present?). This can be done using a UV spectrophotometer (one that can measure absorbance at light wavelengths in the UV range. Procedure 1. Determination of DNA concentration To measure the concentration of DNA in solution, the absorbance is measured (in absorbance units, or au) at 260 nm. Since 50 µg/ml of DNA in solution gives an absorbance of 1 au, the following equation can be used to give the DNA concentration in µg/ml: DNA concentration 50 µg/ml = X µg/ml equation 1 au au at 260 nm EX: the absorbance of a sample of DNA solution at 260 nm is measured as 0.25 au. What is the concentration of DNA in the sample? 50 µg/ml = X µg/ml = 0.25 x 50 µg/ml = 12.5 µg/ml 1 au 0.25 au 2. Determination of DNA purity The test to determine the purity of a DNA sample is a simple calculation of the ratio of absorbance at 260 nm (the wavelength of DNA absorbance) to the absorbance at 280 nm (the wavelength of protein absorbance). This is called a 260/280 reading. DNA purity equation abs at 260nm abs at 280 nm If the 260/280 ratio is approximately 1.8, the DNA sample is considered fairly pure. If it s too high, RNA contamination is suspected. If it s too low, protein contamination is suspected. Your instructor will give you additional instructions during lab and will help you measure the absorbance of your sample at 260 nm and 280 nm on the UV spectrophotometer. After you have recorded the data in your notebook, calculate your DNA concentration and DNA purity and compare with other groups to see how effective your miniprep procedure was at purifying the DNA from the transformed bacterial cells. 93

6 Lab 10 Homework Name: 1. What new traits did the bacteria acquire when they were transformed with the pglo plasmid? (Hint: there are two new traits acquired) 2. If someone gave you a vial of lab stock E. coli, how would you be able to tell if they were ampicillin resistant? What steps would you take to determine this? 3. You are transforming bacterial cells with a plasmid containing a gene that allows the bacteria to digest toxic heavy metals. In the same plasmid, you also insert the betalactamase gene for ampicillin resistance. a) It s the heavy metal detoxification gene that you re interested in. So, what is the purpose of having the beta-lactamase gene on this plasmid? b) You want to calculate the transformation efficiency after performing your experiment. Here is the data you have recorded in your lab notebook: - Your plasmid DNA solution had a concentration of 0.01 µg/ml. - You mixed 15 µl of plasmid DNA solution with 285 µl of transformation solution. - After the transformation procedure, you added 500 µl of LB nutrient broth and incubated the mixture for 10 minutes at 37 C. - You spread 80 µl of the liquid on different LB plates and incubate at 37 C overnight. - The next day, you count 210 colonies on your LB/amp plate. (Your negative control with no plasmid DNA on LB/amp plates had no colonies) 94

7 3. (continued) Given this data, calculate the transformation efficiency. 4. Looking over the steps of the miniprep protocol, describe what is happening in each of the following steps: a) Lysis b) Neutralization c) Binding d) Elution 5. After purifying plasmid DNA from bacterial cells, you measure the absorbance of the DNA solution in a UV spectrophotometer at 260 nm and at 280 nm. The absorbance at 260 nm measures 1.5. The absorbance at 280 nm measures 1.0. a) What is the concentration of the DNA? b) Comment on the purity of the DNA. 95

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