(Solved): Understanding PCR and DNA extraction 0.8% Agarose ...

Understanding PCR and DNA extraction

$0.8\%$ Agarose (GP) \begin{tabular}{l|llllllllll} 1234 & 2 & 5 & 6 & 8 & 9 & 10 & 12 \end{tabular} 5. What do you conclude from the experiment? Did your PCR work? What are the limitations to this experiment? What additional experiments could you do to strengthen your conclusions? If the experiment did not work what are some reasons that this could have occurred. 4. Observe the photograph of the stained gel containing your PCR samples. Orient the photograph with the sample wells at the top. Use the sample gel shown below to help interpret the band(s) in each lane of the gel. a. Locate the lane containing the pBR322/BstNI markers on the left side of the gel. Working from the well, locate the bands corresponding to each restriction fragment: $1857\mathrm{bp},1058\mathrm{bp}$, $929\mathrm{bp},383\mathrm{bp}$, and $121\mathrm{bp}$. The 1058-bp and 929-bp fragments will be very close together or may appear as a single large band. The 121- bp band may be very faint or not visible. b. (Alternatively, use a 100-bp ladder as shown on the right-hand side of the sample gel. These DNA markers increase in size in 100-bp increments starting with the fastest migrating band of $100\mathrm{bp}$. Use the brighter 500-bp and 1000-bp bands as references.) c. Scan across the photograph of your gel. Each of the experimental lanes should contain one prominent band. The PCR products of wild-type and dpy-13 RNAi-treated wild-type worms should align between the 1058-bp and 929-bp fragments of the pBR322/BstNI markers. The PCR product of the dpy-13 mutant worms should run slightly ahead of the 383-bp marker fragment. d. It is common to see a diffuse (fuzzy) band that runs ahead of the 121-bp marker. This is "primer dimer," an artifact of the PCR reaction that results from the primers overlapping one another and amplifying themselves. The presence of primer dimer, in the absence of other bands, confirms that the reaction contained all components necessary for amplification. e. Additional faint bands at other positions on the gel occur when the primers bind to chromosomal loci other than dpy-13, giving rise to "nonspecific" amplification products. How could you modify the PCR reaction conditions to attempt to eliminate these products? 1. Describe the appearance of each of the three types of worms from which DNA was isolated in Part III: a. wild-type worms on NGM-lite plate b. dpy-13 RNAi-treated wild-type worms on NGM-lite/amp+IPTG plate c. dpy-13 mutant worms on NGM-lite plate What does this suggest about RNAi and how it impacts the phenotype of $C$. elegans? 2. Describe the purpose of each of the following steps of DNA isolation (Part III): a. Freezing b. Incubating with proteinase $K$ c. Boiling at $95^{?}\mathrm{C}$ 3. What are the three steps of PCR and their purposes? Which of these steps is when the primers bind to the DNA and varies in temperature depending on the primer pair? 1. TRANSFEA WILD-TYPE AND dP -13C. elegans TO OPSO-SEEDED NGM-LITE PLATES IV. ANA To feed worm strains, it is easier to transfer a chunk of worm-filled agar from a well-grown plate to a new plate rather than pick individual worms. The dnv-13 strain is a control. 1. Use a black pen to label the bottom of an OP50-seeded plate with the date and "wild-type." 2. Use a black pen to label the bottom of another OP50-seeded plate with the date and "dpy-13." 3. Sterilize a metal spatula or forceps. Dip the end of the implement into the ethanol beaker, and briefly pass it through a Bunsen flame to ignite the alcohol. Allow alcohol to burn off away from the Bunsen flame; the implement will become too hot if left in the flame. (Sterilization prevents cross-contamination with different $C$. elegans strains and non-OP50 bacteria.) CAUTION Be extremely careful not to ignite the ethanol in the beaker. Do not panic if the ethanol is accidentally ignited. Cover the beaker with a petri dish lid or other non-flammable cover to cut off oxygen and rapidly extinguish the fire. 4. Use the sterilized implement to cut a 1-cm (about $3/3$ in.) square chunk of agar with worms from the wild-type starter plate. 5. Transfer the chunk to the OP50-seeded plate labeled "wild-type," and place it face down on the agar. (This allows worms to quickly crawl into the bacteria on the new plate.) 6. Use the same procedure to transfer a chunk of agar with worms from the dpy-13 starter plate to the OP50-seeded plate labeled "dpy-13." 7. Incubate the plates upside down at $20^{?}\mathrm{C}$ for 48 hours. Choose a place where the plate will not be disturbed. This will give the newly transferred worms time to grow. 1. TRANSFER WILD-TYPE AND dpy-13 C. elegans TO OP50-SEEDED NGM-LITE PLATES 1. Use a black pen to label the bottom of an OP50-seeded plate with the date and "wild-type." 2. Use a black pen to label the bottom of an OP50-seeded plate with the date and "dpy-13." 3. Use a black pen to label the bottom of the plate seeded with the dpy13 RNAi feeding strain with the date and "wild-type." 4. Pick and transfer five L4-stage worms (see illustration below) from your plate of wild-type worms to the OP50-seeded plate labeled "wild-type." 5. Confirm that the transferred worms are the correct stage and were not injured or killed during the picking process. 6. Identify any eggs or young larvae that may have been accidentally transferred. Pick them off the plate, and flame them in a Bunsen burner. 7. Use the same method to move five $L4$ wild-type worms to the plate seeded with the $dpy?13$ RNAi feeding strain. 8. Use the same method to move five L4 dpy-13 worms to the OP50seeded plate labeled "dpy-13." 9. Incubate the plates upside down at $20^{?}\mathrm{C}$. Choose a place where the plates will not be disturbed. 10. The day after transferring, check that your worms are still healthy. Note any dead worms and pick them off the plate. 11. On the third day after transferring, pick the remaining adult worms III. Isolate DNA from C. elegans T. Label three PCR tubes with your group number. Label one tube "W" (wild type), one "R" (RNAi), and one "D" (deletion mutant). 2. Use a micropipet with a fresh tip to add $10?\mathrm{L}$ of lysis buffer to each tube. 3. Observe the wild-type, $dpy?13$, and $dpy?13$ RNAi-treated wild-type worms under a dissecting microscope. Note differences in the length and width of the worms. 4. Use a sterile toothpick to transfer four or five adult wild-type worms to the "W" tube. Swish the tip directly in the lysis buffer to dislodge the worms from the toothpick. 5. Observe the PCR tube under a stereomicroscope. Verify that at least three worms are alive and writhing in the buffer. If you have trouble seeing the worms, flick the tube or momentarily centrifuge the tube to ensure that the worms are at the bottom. 6. Pick and transfer four or five $dpy?13$ RNAi-treated wild-type worms to the " $R$ " tube, and verify that at least three live worms are in the lysis buffer. 7. Pick and transfer four or five $dpy?13$ worms to the "D" tube, and verify that at least three live worms are in the lysis buffer. 8. Fit each of your PCR tubes into one or two adaptors. PCR tubes are "nested" into sequentially larger adaptors: a 0.2-mL tube, within a 0.5$\mathrm{mL}$ tube, within a $1.5?\mathrm{mL}$ tube. 9. Place your tubes in a balanced configuration in a microcentrifuge, and spin for 5-10 seconds at full speed. This will pellet the worms. 10. Place your tubes in liquid nitrogen, on dry ice, or in a freezer until the liquid in the tubes is frozen solid. (Freeze-thawing cracks the tough outer cuticle of the worm.) 11. Place your $PCR$ tubes, along with other student samples, in a thermal cycler that has been programmed for one cycle of the following profile. The profile may be linked to a $4^{?}\mathrm{C}$ hold program. Incubating step: $65^{?}\mathrm{C}90$ minutes Boiling step: $95^{?}\mathrm{C}15$ minutes (Proteinase $K$ in the lysis buffer digests protein in the cuticle and helps to liberate individual cells by digesting protein fibers of the extracellular matrix that bind cells together. It also inactivates cellular proteins, including DNases that interfere with PCR amplification. The near-boiling temperature lyses individual cells and inactivates the proteinase $K$.) 12. Store your sample on ice or at $?20^{?}\mathrm{C}$ until you are ready to continue with Part IV. IV. Amplify DNA by PCR 1. Obtain three tubes that each contain a Ready-To-Go ${}^{\text{TM }}$ PCR Bead. Label each tube with your group number. Label one tube "W" (wild-type), one " $R$ " (RNAi), and one " $D$ " (deletion mutant). 2. Use a micropipet with a fresh tip to add $22.5?\mathrm{L}$ of $dpy?13$ primer/loading dye mix to each tube. Allow the bead to dissolve for a minute or so. 3. Use a micropipet with a fresh tip to add $2.5?\mathrm{L}$ of worm DNA from Part III to the appropriate tubes. a. Add the DNA directly into primer/loading dye mix. b. Ensure that no worm DNA remains in the tip after pipetting. c. Use a fresh tip for each reaction. 4. If your thermal cycler does not have a heated lid: Prior to thermal cycling, you must add a drop of mineral oil on top of your PCR reaction. Be careful not to touch the tip of the oil-dispensing pipet to the tube or reaction, or the oil will be contaminated with your sample. 5. Store your samples on ice until your class is ready to begin thermal cycling. 6. Place your PCR tubes, along with other student samples, in a thermal cycler that has been programmed for 30 cycles of the following profile. The profile may be linked to a $4^{?}\mathrm{C}$ hold program after the 30 cycles are completed. 7. After cycling, store the amplified DNA on ice or at $?20^{?}\mathrm{C}$ until you are ready to continue with Part $V$. V. Analyze PCR Products by Gel Electrophoresis 1. Seal the ends of the gel-casting tray with masking tape or sealing device. 2. Insert a well-forming comb at one end of the tray. 3. Pour $1\%$ TBE agarose solution into the tray to a depth that covers about one-half the height of the open teeth of the comb. 4. Allow the gel to solidify completely. This takes approximately 20 minutes. 5. Remove the tape or sealing device, and place the gel into the electrophoresis chamber. 6. Add enough $1\times$ TBE buffer to just cover the surface of the gel. 7. Carefully remove the comb, and add additional $1\times$ TBE buffer to just cover and fill in wells, creating a smooth buffer surface. marker into the far left lane of the gel. (Alternatively, use $0.5?\mathrm{g}$ of 100-bp ladder.) 9. Use a micropipet with a fresh tip to add all of each sample/loading dye mixture into a different lane of the gel, according to the following diagram. (If you used mineral oil during $PCR$, pierce your pipet tip through the layer of mineral oil to withdraw the PCR sample and leave the mineral oil behind in the original tube.) 10. Run the gel at $130\mathrm{V}$ for approximately 30 minutes. Adequate separation will have occurred when the cresol red and bromophenol blue dye front has moved at least $50\mathrm{mm}$ from the wells. Do not run the dye front to the end of the gel. 11. Stain the gel in ethidium bromide (EtBr) or CarolinaBLU: a. For ethidium bromide, soak the gels for at least 10-15 minutes. Decant stain back into the storage container for reuse, and rinse the gel in tap water. Use gloves when handling ethidium bromide solution, a stained gel, or anything that has ethidium bromide on it. Ethidium bromide is a known mutagen and care should be taken when using and disposing of it. b. For CarolinaBLU staining, follow directions in the Instructor section on page 31. 12. View the gel using a transilluminator, and photograph it.