CSU Biol 5515 PCR Protocol KAUR

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7020.10Hd Z H31ldVHO Cloning and Sequencing Explorer Series Protocol Overview The strategy for this experiment uses . nested PCR to amplify portions of the Cloning the GAPC gene GAPC gene from gDNA of the plant of interest.Three control reactions will also Identify and extract gDNA from plants be set up. A negative control will be run e Amplify region of GAPC gene using PCR with sterile water instead of gDNA to ¢ Assess the results of PCR test for contamination, and two positive e Purify the PCR product controls will be run with a plasmid and Arabidopsis gDNA. The plasmid control ensures the PCR reagents and thermal cycler are functioning. The Isolate plasmid from the bacteria pGAP control plasmid contains the Sequence DNA GAPC target region from Arabidopsis e Perform bioinformatics analysis of the cloned gene and shoud yield an intense single band around 1 kb. The second positive control with Arabidopsis gDNA more closely matches your extracted plant gDNA. The gDNA control provides a more representative PCR result since amplification of genomic DNA is much less efficient than plasmid DNA. The band intensity for this reaction will be lower than for the plasmid and may also yield multiple bands representative of the four GAPC genes in Arabidopsis. The gDNA control also provides a control template for the nested PCR, and products from this control nested PCR may be used to continue the lab in the event that the plant samples did not amplify. In the initial round of PCR, a set of blue primers using degenerate (ess specific) sequences will amplify the GAPC gene from the gDNA. Then, in the second round of PCR (the nested PCR), a more specific set of yellow primers will amplify GAPC from the initial PCR products. It is very important not to reverse the order in which the primers are used or to mix the two primer sets together in the PCR reactions. ¢ Ligate PCR product into a plasmid vector e Transform bacteria with the plasmid Before performing the nested PCR, the primers that were not incorporated into PCR product in the first round must be removed so that they do not amplify target DNA in this round of PCR. To remove the primers, exonuclease |, an enzyme that specifically digests single-stranded DNA such as primers but not double-stranded DNA such as the template, wil'be added to the PCR products from the first round. After exonuclease | digests the primers, the enzyme must be inactivated to prevent the exonuclease from digesting the nested primers that will be added for the next round of PCR. Following exonuclease | treatment and inactivation of the enzyme, the PCR products from the gDNA templates generated in the first round of PCR need to be diluted. The PCR products from the initial round contain a high proportion of GAPC-like sequences relative to the total amount of gDNA. By diluting the gDNA, it is even less likely that the gDNA will be a template for contaminating PCR products when using the nested primers in the next round of PCR. Because the complexity of the pool of available DNA templates has been greatly reduced, nested PCR is very efficient. As each PCR reaction takes approximately 3—4 hours to run, it is most practical to run the PCR reactions on separate days. Since the reagents used in these experiments function optimally when prepared fresh, it is highly-recommended that the reagents be prepared just prior to setting up the reactions. [=][eF'=7:1») 60 GAPDH PCR

700010Hd 2 H31ldVHO Cloning and Sequencing Explorer Series oo Preparation for Initial PCR (First-Round PCR) T Plan the first round of PCR. You will perform one initial PCR for each of the two plant gDNA samples you have extracted, two positive controls, one using control gDNA and the other using pGAP plasmid DNA, and one negative control with sterile water instead of DNA, for a total of 5 PCR reactions. Use the table below to record the label on each PCR tube, the DNA template, and the primers used to amplify the DNA. Prepare a master mix. (Note: This mix should be prepared no more than 30 minutes prior to performing PCR. Your instructor may have prepared it just prior to the lab.) A master mix is a mixture of all the reagents required for PCR except the template DNA. Making a single mixture reduces potential pipetting errors, and increases the consistency between PCR samples. Tube Label Template Primers Bio-Rad'’s 2x master mix is provided as a 2x (double strength) colorless reagent; when mixed with an equal amount of DNA template, all components are at optimal concentrations in the final reaction. Bio-Rad 2x master mix contains 7ag DNA polymerase, dNTPs, buffer, and salt, but does not contain primers. It is necessary to add primers to Bio-Rad 2x master mix to form a complete 2x master mix with initial primers — “2x MMIP". Consider why commercial master mixes are not sold with primers added to them. Give one reason. Each PCR reaction volume is 40 pl. Thus, each reaction requires 20 pl of 2x MMIP (Bio-Rad 2x master mix with blue initial primers) plus 20 pl of DNA template. The amount of 2x MMIP to prepare is calculated by multiplying the number of reactions plus one (to allow for pipetting errors) by the volume of 2x MMIP required for each reaction (20-pl). Referring to your table, calculate how much 2x MMIP is required. Volume of 2x MMIP required = (# PCR reactions + 1) x 20 pl = ul For the initial round of PCR, blue initial primers will be used. These primers are designed for a section of DNA bracketing the target sequence. The initial primers are supplied at a 100 pM concentration. For this PCR, the concentration of primers in the 2x MMIP should be 2 M. Calculate the volume of initial primers required in the 2x MMIP. Remember the formula MV, = M,V,, where: M, = Required concentration of primers (uM) V, = Required volume of 2x MMIP (ul) M, = Given concentration of pimers (UM)), V, = Required volume of primers (pl) Required volume of intial primers (V,,): ul [=][eX=7:1p] 62 GAPDH PCR

7000.104Hd 2 H31ldVHO Cloning and Sequencing Explorer Series % Preparation for Initial PCR (First-Round PCR) 1. Plan the first round of PCR. You will perform one initial PCR for each of the two plant gDNA samples you have extracted, two positive controls, one using control gDNA and the other using pGAP plasmid DNA, and one negative control with sterile water instead of DNA, for a total of 5 PCR reactions. Use the table below to record the label on each PCR tube, the DNA template, and the primers used to amplify the DNA. Prepare a master mix. (Note: This mix should be prepared no more than 30 minutes prior to performing PCR. Your instructor may have prepared it just prior to the lab.) A master mix is a mixture of all the reagents required for PCR except the template DNA. Making a single mixture reduces potential pipetting errors, and increases the consistency between PCR samples. Tube Label Template Primers Bio-Rad'’s 2x master mix is provided as a 2x (double strength) colorless reagent; when mixed with an equal amount of DNA template, all components are at optimal concentrations in the final reaction. Bio-Rad 2x master mix contains 7ag DNA polymerase, dNTPs, buffer, and salt, but does not contain primers. It is necessary to add primers to Bio-Rad 2x master mix to form a complete 2x master mix with initial primers — “2x MMIP”. Consider why commercial master mixes are not sold with primers added to them. Give one reason. Each PCR reaction volume is 40 pl. Thus, each reaction requires 20 pl of 2x MMIP (Bio-Rad 2x master mix with blue initial primers) plus 20 pl of DNA template. The amount of 2x MMIP to prepare is calculated by multiplying the number of reactions plus one (to allow for pipetting errors) by the volume of 2x MMIP required for each reaction (20-pl). Referring to your table, calculate how much 2x MMIP is required. Volume of 2x MMIP required = (# PCR reactions + 1) x 20 pl = pl For the initial round of PCR, blue initial primers will be used. These primers are designed for a section of DNA bracketing the target sequence. The initial primers are supplied at a 100 pM concentration. For this PCR, the concentration of primers in the 2x MMIP should be 2 M. Calculate the volume of initial primers required in the 2x MMIP. Remember the formula M,V, = M,V,, where: M, = Required concentration of primers (UM) V, = Required volume of 2x MMIP (ul) M, = Given concentration of pimers (UM)), V, = Required volume of primers (pl) Required volume of intial primers (V,,): pl [=][oX=7:1p] 62 GAPDH PCR

Cloning and Sequencing Explorer Series Label a microcentrifuge tube 2x MMIP. No more than 30 min before use, add the calculated volume of initial primers to the required volume of Bio-Rad 2x master mix in a labeled tube. Mix well by pipetting up and down several times or vortexing. If a microcentrifuge is available, spin tube briefly to collect the contents at the bottom of the tube. Keep on ice. Experimental Procedure for Initial PCR 1. Referring to your table, label your PCR tubes with your initials and the tube label. 2. Place each PCR tube into a tube adaptor and cap each tube. Place the adaptor tube with PCR 070 adaptor 3. Ensure all the reagents are thoroughly mixed, especially the gDNA. Mix:tubes containing reagents thoroughly by vortexing or flicking to ensure the gDNA is homogeneously distributed. Before opening the tubes, spin in a microcentrifuge for 5-10 seconds to force contents to the bottom of the tube (to prevent contamination). Each PCR needs to be set up with the following reagents: Pipet 20 pl of 2x MMIP into each PCR tube. Add 15 pl of sterile water to each tube. Referring to your initial PCR plan, use a fresh pipet tip to add 5 pl of the appropriate DNA template to each tube and gently pipet up and down to mix reagents. Use a fresh filter tip each time. Recap tubes tightly to prevent any evaporation during PCR. Reagent Amount Blue master mix (2x MMIP) 20 ’ Sterile water 15 DNA template or negative 5yl control Total 40 ul GAPDH PCR 63 [=][e X =7:Tp) CHAPTER 2 PROTOCOL

7000.10Hd € H31dVHO Cloning and Sequencing Explorer Series 7. When your instructor tells you to do so, place your.PCR tubes into the thermal cycler. The PCR reaction will run for the next several hours using the following Initial GAPDH PCR program: Initial denaturation: 95°C for 5 min Then 40 cycles of: Denaturation: 95°C for 1 min Annealing: 52°C for 1 min Extension: 72°C for 2 min Final extension: 72°C for 6 min Hold: 15°C hold (=) 8. Store PCR products at 4°C for up to 2 weeks and at -20°C long term. Note: Store any remaining gDNA at -20°C. (Optional) Analyze PCR products by agarose gel electrophoresis. This can be performed on the same gel used to analyze the nested PCR products (Chapter 3, Electrophoresis). Prepare agarose gels to analyze the results of your experiment. You will need 1% agarose gels with the sufficient number of wells for each of your samples plus an additional well for your molecular size marker. See Appendix A for detailed instructions on preparing agarose gels. Note: Do not add loading dye directly to initial PCR reactions — the loading dye can inhibit the next round of PCR. Refer to Chapter 3, Electrophoresis for instructions on using electrophoresis to analyze PCR products. The plasmid control should yield a visible band of around 1 kb. It is relatively common for plant gDNA to not yield a visible band during the initial round of PCR and yet be amplified after the nested round of PCR. Results Analysis for Initial PCR Following electrophoresis, consider the following questions regarding your controls and samples: 1. Did the negative control generate a PCR product? If yes, what size were the DNA band(s) and what does this mean for the experiment? If no, what does this mean for the experiment? 2. Did the pGAP plasmid génerate a®PCR product? If yes, what size is the DNA band and what does this mean for the experiment? If no, what does this mean for the experiment? BIO RAD 64 | GAPDH PCR

Cloning and Sequencing Explorer Series Did the control gDNA generate a PCR product? If yes, what size DNA band(s) and what does this mean for the experiment? If no, what does this mean for the experiment? Did your plant gDNA generate PCR products? If yes, what size DNA band(s) and what does this mean for the experiment? If no, what does this mean for the experiment? Plant 1: Plant 2: Preparation for Nested PCR (Second-Round PCR) s Plan the nested PCR experiment. Three nested PCRs will be set up, using as a template the CHAPTER 2 PROTOCOL PCR products of each gDNA sample amplified in the initial round of PCR: the PCR product from the control Arabidopsis gDNA and the two PCR products from the newly extracted plant gDNAs. In addition, two control reactions will be set up with the nested PCR primers: pGAP plasmid as a positive control and sterile water as a negatlve control. Complete the table below to record the label for each nested PCR tube, the DNA template, the dnlutlon factor, and the primers used. Tube Label Template Dilution Factor Primers GAPDH PCR 65

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