Modifications to EXPERIMENTS 21 and 24: PCR and Molecular Cloning This experiment was designed by Dylan Dodd, based on research completed in Dr. Isaac Cann s lab*, with modifications and editing of content by Dr. Grabner. Dr. Grabner would like to acknowledge the significant contribution of time and effort that this work represents. *Dodd D, Kiyonari S, Mackie R, and Cann I. 2010. Functional diversity of four glycoside hydrolase family 3 enzymes from the rumen bacterium, Prevotella bryantii B 1 4. J. Bacteriol. 192, 2335. STUDENTS SHOULD USE A P-2 MICROPIPETTOR FOR ALL VOLUMES 2 µl. Day 1: PCR amplification of a β-xylosidase gene. 1. Set up the following reaction in the specified order in a labeled 0.5-ml sterile PCR tube. Instructors will add polymerase enzymes. Amplification of β-xylosidase 41.5 µl sterile water 5 µl 10X PicoMaxx Buffer 1 µl 10 mm dntp Mix 1 µl 50 ng/µl Prevotella bryantii genomic DNA 1 µl 25 µm oligonucleotide primer mix 0.5 µl PicoMaxx enzyme 2. Place the tube in the PCR Minicycler, which will use the following PCR protocol: PicoMaxx PCR Protocol Initial 95 C 2 min Denature 95 C 40 sec 35 Cycles Anneal 55 C 30 sec Elongate 72 C 3 min Final 72 C 10 min Last 4 C α 1
Day 2: Digestion and Gel Purification of PCR Products *Students will need to come to lab early in order to shorten the length of time in lab on Day 2. Only one group member needs to be present for these steps. Students in both sections will need to set up the restriction digest at 12:00 pm. 1. Set up the following reaction in a labeled 0.7-mL sterile microfuge tube. Instructors will add restriction enzymes. Please note that this is a digestion of the PCR product. 2. Incubate in a 37 C water bath for ~2 hr. 3. Reserve the uncut sample for your gel. Digestion of PCR product* 12 µl sterile water 3 µl 10X NEB Buffer 4 3 µl 10X BSA 10 µl PCR Sample 1 µl NdeI restriction enzyme 1 µl XhoI restriction enzyme 4. While the digests are incubating, set up a 1% agarose gel two groups will share one gel. Do NOT move the gel from the designated area. Carefully remove the cast gel from the casting tray and transfer it to the submarine unit. Pour 250 ml of running buffer (1X TBE) over the gel. The gel should be completely covered with buffer. 4. Add 6 µl of 6X loading dye to the PCR sample after digestion is complete. 5. Make your uncut gel sample by combining 10 µl of uncut PCR sample, 20 µl H 2 O and 6 µl 6X loading dye. 6. Load the entire volume of the above samples into the gel wells according to the following order. Also load 10 µl of the DNA ladder in lane 5 (middle of the gel). Lane 1: Nothing Lane 2: Uncut PCR, Group 1 Lane 3: Digested PCR, Group 1 Lane 4: Nothing Lane 5: DNA ladder Lane 6: Nothing Lane 7: Uncut PCR, Group 2 Lane 8: Digested PCR, Group 2 7. Attach the negative electrode (black) to the well side of the chamber and the positive electrode (red) to the other side of the chamber. Remember that the DNA is negatively charged and will 2
migrate toward the positive electrode. Run the gel at 120 V constant voltage until the first dye front has migrated 2/3 of the gel length (~30 minutes). 8. Turn off the power to the electrophoresis chamber, lift the gel tray out of the box, and take the gel to the EtBr supplemented water bath. Allow the gel to stain ~15 min. After staining, destain the gel for 10 min in a water bath. Then, image the gel (TA will assist you). MESSES WILL NOT BE TOLERATED, PLEASE CLEAN UP AFTER YOURSELF! 9. While the gel is on the UV box, take a picture of the gel. Gloves and goggles are mandatory when using UV light. Try to minimize the amount of time that your gel is exposed to UV light because it may cause adjacent thymine bases to dimerize, which leads to mutations in the DNA. 10. Using your picture, estimate the sizes of the DNA fragments of your two samples by comparing them to the DNA ladder. Record the estimated sizes in your laboratory notebook. Using the long-wavelength setting on a handheld UV lamp and a razor blade, excise only the band for the Xyl3B gene in the digested PCR reaction. Try to keep the size of the gel slice as small as possible without leaving DNA behind in the gel. As a general rule of thumb, the smaller the gel slice obtained, the more efficient the gel purification will be! 11. Add the gel slice into a pre-weighed 1.5-ml microfuge tube. Weigh the tube with the gel slice in order to determine the gel mass. Ideally, the gel mass should be 300 mg. 12. Add 500 µl of DF Buffer to the gel slice and mix by vortex. 13. Incubate at 55 C for 15 min (or until the gel slice has completely dissolved). To help dissolve the gel, mix by inverting the tube every 2 3 min during the incubation. Be sure to solubilize the agarose completely. Cool the dissolved sample mixture to room temperature. 14. Place the DF Column into a 2-ml Collection Tube. To bind DNA, apply the sample to the DF Column without touching the filter and centrifuge at maximum speed for 30 sec. The maximum volume of the column reservoir is 800 µl. For sample volumes of more than 800 µl, simply load the additional volume and spin again. 15. Discard flow-through and place the DF Column back in the same collection tube. Collection tubes are reused to reduce plastic waste. 16. Add 400 µl of W1 Buffer to the DF Column and centrifuge at maximum speed for 30 sec. Discard the flow-through and place the DF Column back in the same collection tube. 17. Add 600 µl of Wash Buffer (with EtOH) to the DF Column and let stand for 1 min. Centrifuge at maximum speed for 30 sec and discard the flow-through. 18. Place the DF Column back in the collection tube and centrifuge for an additional 3 min to dry the column matrix. IMPORTANT: Residual ethanol from Wash Buffer will not be completely removed unless the flow-through is discarded before this additional centrifugation. It is important to remove the residual ethanol as it can inhibit downstream reactions. 19. Transfer the dried DF Column into a clean 1.5-ml microfuge tube. 3
20. To elute DNA, add 20 µl of 37 C prewarmed Elution Buffer (10 mm Tris-HCl, ph 8.5) to the center of the DF Column matrix and let the column stand for 2-5 min. Centrifuge for 2 min at maximum speed to elute the purified DNA. IMPORTANT: Ensure that the elution buffer is dispensed directly onto the column matrix for complete elution of bound DNA. 21. Carefully mix the following together in a sterile, 0.2 ml PCR tube. Instructors will add the enzyme: Ligation Ligation Control 5 µl digested PCR sample 5 µl sterile water 2 µl pre-digested pet15b vector 2 µl pre-digested pet15b vector 2 µl T4 DNA 5X ligase buffer 2 µl T4 DNA 5X ligase buffer 1 µl T4 DNA Ligase 1 µl T4 DNA Ligase 22. Incubate the reaction at 15 C overnight. Store the rest of your DNA sample (unused) at 4 C. Day 3: Transformation of DH5α E. coli Cloning Cells 1. Obtain a microfuge tube from the instructor containing 160 µl of freshly thawed DH5α competent cells. Keep these cells on ice at all times! 2. Very gently, mix the cells by flicking the tube gently with your finger, then add 40 µl of these cells to each of 4 pre-chilled 15-ml polypropylene culture tubes. Label the tubes 1 to 4 and keep them on ice at all times. 3. To tube 1, add 1 µl of the ligation mixture. To tube 2, add 1 µl of pet15b vector (0.1 ng/µl). To tube 3, add 1 µl of 1X ligation buffer (no DNA). To tube 4, add 1 µl of ligation control reaction. Very gently, mix the reactions by flicking the tubes as before (step 2). 4. Incubate the cells with these samples on ice for 15 min, swirling gently every 2 min. Quickly remove the tubes from the ice and place them in a 42 C water bath. Incubate the tubes at 42 C for exactly 45 sec. Quickly remove the tubes from the water bath and incubate them again on ice for 2 min. During the heat shock, the competent E. coli cells will take up the intact plasmid DNA. The cells are very compromised at this point and will die if the heat shock is too long (> 1 min). The incubation on ice prevents the cells from dying after heat shock. 5. Add 1.0 ml of L-broth each tube. Incubate the cells at 37 C with gentle shaking for 1 h, during which time the cells will recover from the heat shock. 6. Obtain 6 LB-agar plates containing ampicillin (100 µg/ml). 7. Using L-broth, prepare 1:10 dilutions of transformation cultures #1 and #2 in 1.5-ml microfuge tubes to a final volume of 0.5 ml. 8. Remove 200-µl aliquots from undiluted culture tubes 1-4 and place on 4 separate LB/Amp plates. Remove 200-µl aliquots from the two diluted culture samples and place on 2 separate LB/Amp 4
plates. Be sure to label around the edge of each plate with the name of the antibiotic, as well as the identity and dilution of the transformation mixture added. 9. Using sterile technique (demonstrated by TA), use a sterile bent glass rod, EtOH, and flame to spread the bacteria evenly over the surface of each plate. Allow the surface of the plates to dry for 5 min, invert the plates (agar side up), and place them in the 37 C incubator overnight. Extra Day: Growth of Transformed Cells 1. Retrieve your 6 plates from the 37 C incubator. Obtain 3 culture tubes and add 3 ml of LB broth. Supplement with 3 µl of 100 mg/ml ampicillin stock (final concentration is 100 µg/ml). Label the tubes with your initials and number 1 to 3. 2. Using a sterile toothpick, select a portion of a single colony that is present on either of the plates containing cells from transformation tube 1. Remove the cap, and drop the toothpick (bacteria end down) into culture tube 1. Repeat this step for the plates containing cells from transformation tubes 2 and 4. 3. Incubate the cultures at 37 C with shaking overnight. These strains will be used on Day 4 to isolate plasmid DNA. 4. Count the number of colonies on the plate containing cells from transformation tube 2. Why was this control experiment performed? Describe what a low number of colonies on this plate would indicate, as well as possible causes for this result. 5. Count the number of colonies on the plate containing cells from transformation tube 3. Why was this control experiment performed? Describe what a high number of colonies on this plate would indicate, as well as possible causes for this result. 6. Count the number of colonies on the plate containing cells from transformation tube 4. Why was this control experiment performed? Describe what a high number of colonies on this plate would indicate, as well as possible causes for this result. 7. Calculate the transformation efficiency as the number of transformants per ng of DNA, using your control plasmid transformation sample (#2). Keep in mind that each colony derives from a single cell (transformant), and that you need to determine the # of transformants per ng DNA plated. 8. Wrap each of your plates with Parafilm and store them agar-side up at 4 C (cold room). Day 4: Isolation of Plasmid DNA, Restriction Digestion, and Agarose Gel Electrophoresis 1. Remove the 3 culture tubes from the 37 C shaker. Each group will prep all of the 3 cultures inoculated the previous day. Add 1.5 ml of the three cultures to 3 separate microfuge tubes labeled 1 through 3. Cap each tube and centrifuge for 2 min at room temperature. Remove and discard the supernatant. 5
2. Add the remaining 1.5 ml of each culture to the appropriate microfuge tube. Cap each tube and centrifuge for 2 min at room temperature. Remove and discard the supernatant. 3. Thoroughly re-suspend each cell pellet in 200 µl of PD1 Buffer by vortex or pipetting. 4. Add 200 µl of PD2 Buffer to each tube and mix gently by inverting the tube 10 times. DO NOT vortex to avoid shearing the DNA. Let stand at room temperature for NO MORE than 5 min. This step ensures that lysis is complete. 5. Add 300 µl of PD3 Buffer to each tube, cap the tubes, and mix immediately by inverting the tubes 10 times. DO NOT vortex. Remember, this must be added no later than 5 min after the addition of the PD2 Buffer. Centrifuge at maximum speed for 3 min. 6. Place a PD Column into each of three 2-ml Collection Tubes. Transfer the supernatant of each tube from Step 5 to one of the columns, leaving behind the white precipitate. Centrifuge at maximum speed for 30 sec. 7. Discard the flow-through from each of the bottom tubes and replace the column back into the Collection Tube. Add 400 µl of W1 Buffer to each column. Centrifuge at maximum speed for 30 sec. 8. Discard the flow-through from each of the bottom tubes and replace the column back into the Collection Tube. Add 600 µl of Wash Buffer (with EtOH) to each column. Centrifuge at maximum speed for 30 sec. 9. Discard the flow-through from each of the bottom tubes and replace the column back into the Collection Tube. Centrifuge the empty columns at maximum speed for 3 min to dry the column matrix. Remember, the goal here is to remove all traces of EtOH. 10. Transfer each of the dried columns to clean microfuge tubes. Add 50 µl of Elution Buffer to the center of each column. Incubate at room temperature for 2-5 min. Centrifuge at maximum speed for 2 min, making sure the tube caps are toward the center of the rotor. Be sure to put the lid on the rotor to keep from shearing off the tube caps. 11. Set up the following restriction digests for each of your 3 plasmid samples in 0.7-ml microfuge tubes according to the table below. Be sure to label the microfuge tubes with the appropriate culture sample and digest. You should have 9 digest tubes. XhoI/XbaI Double Digest HincII Digest NdeI Digest 8 µl DNA prep 8 µl DNA prep 8 µl DNA prep 2 µl 10X Buffer 4 2 µl 10X Buffer 3 2 µl 10X Buffer 4 2 µl 10X BSA 2 µl 10X BSA 1 µl 1:4 NdeI 1 µl 1:4 XhoI 1 µl 1:4 HincII 8 µl sterile water 1 µl 1:4 XbaI 7 µl sterile water 6 µl sterile water 12. Incubate the digests at 37 C for 1 hr 30 min. 6
13. While the digests are incubating, set up a 1% agarose gel one group will use each gel. Do NOT move the gel from the designated area. Carefully remove the cast gel from the casting tray and transfer it to the submarine unit. Pour 250 ml of running buffer (1X TBE) over the gel. The gel should be completely covered with buffer. 14. When the digests are complete, centrifuge the tubes at maximum speed for 5 sec in order to spin down any condensate. Add 4 µl of loading dye to each digest. Also set up three tubes of uncut plasmid (one of each sample) and add the following: 8 µl of undigested plasmid from your minipreps, 12 µl of sterile water and 4 µl of loading dye. 15. Load 10 µl of each sample into the gel wells according to the following order. Also load 10 µl of the DNA ladder in lane 4 (middle of the gel). Lane 1: Nothing Lane 2: DNA Ladder Lane 3: Uncut plasmid #1 Lane 4: XhoI/XbaI double digest #1 Lane 5: HincII digest #1 Lane 6: NdeI digest #1 Lane 7: Uncut plasmid #2 Lane 8: XhoI/XbaI double digest #2 Lane 9: HincII digest #2 Lane 10: NdeI digest #2 Lane 11: Uncut plasmid #3 Lane 12: XhoI/XbaI double digest #3 Lane 13: HincII digest #3 Lane 14: NdeI digest #3 Lane 15: Nothing 16. Attach the negative electrode (black) to the well side of the chamber and the positive electrode (red) to the other side of the chamber. Remember that the DNA is negatively charged and will migrate toward the positive electrode. 17. Run the gel at 100 V constant voltage until the first dye front has migrated 2/3 of the gel length. 18. Turn off the power, lift the gel tray out of the box, and EtBr stain/destain the gel as done on Day 2. Take a picture of your gel for your lab notebook. Be sure to wear UV-protective shielding while viewing your gel. Clearly label the samples in each lane and the MWs found on the ladder. 19. Estimate the sizes of the DNA fragments of your samples by comparing them to the DNA ladder. 20. Have you successfully constructed the desired pet15b/xyl3b recombinant plasmid? Explain your answer in terms of what size DNA fragments resulted from each digest and your knowledge of the composition of the parent plasmid, and the insert gene used in this experiment. Prepare a restriction map of the newly constructed recombinant plasmid. The map should include the total number of base pairs in the plasmid, and the identity and position of the following restriction sites in the plasmid: XhoI, XbaI, NdeI, and HincII. 7
21. If you do not think that you have isolated the desired pet15b/xyl3b plasmid, prepare a map of the plasmid that you think you have isolated. Support your map with an explanation of how the digestion of this plasmid with the restriction enzymes used would produce the results that you obtained. 8