Q1 Instructions: 1. Use the stock and target concentrations from Table 1 and the indicated total reaction volume to calculate the volume of components 2-6 for a single reaction. Then, calculate the volume of water needed so that your total reaction volume matches the amount indicated in the table. 2. Multiply the single reaction volumes of components 1-6 by the number of reactions you will set up to get the final recipe for your mastermix. Notes: - The square braces, [ ], indicate a value that you need to calculate. - All your values should be calculated as if the units are µl, though you will enter only the numerical value as your answer. Keep up to 2 decimal places if needed. - Values in bold are different on each students' worksheet. Table 1 Mastermix recipe to prepare 15 PCR reactions your genotyping experiment Component | Stock concentration | Target concentration | µl for single rxn | µl for mastermix 1. dH2O | na | na | [ ] | [ ] 2. PCR buffer | 5x | 1x | [ ] | [ ] 3. MgCl2 | 25mM | 2mM | [ ] | [ ] 4. Primer mix | 20µM | 0.2µM | [ ] | [ ] 5. dNTP's | 20mM | 0.2mM | [ ] | [ ] 6. Taq polymerase* | 20 units*/µl | 1 unit | [ ] | [ ] 7. Template DNA | na | na | 2µl | (omit from mix) Total volume: 50µl * "Units" are a measure of enzyme activity. Calculate the volume of Taq stock solution to give 1 unit per reaction. This will be a different formula than for the other ingredients.
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6 grams of Aguero's in 200 ml, we can calculate the amount in 100 ml: 1.6 g / 2 = 0.8 g ** Show more…
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a) Each PCR reaction will need two PCR primers. Each primer will be given to you as a 5M stock solution. Your final PCR reaction must contain 0.2 M of each primer. The final total volume for the PCR must be 25 L. How much of each primer will you need to add? b) You need to add 2X PCR mix. This mix contains the Taq DNA polymerase, Mg++, buffer and dNTPs required for the reaction. The PCR mix is supplied at a 2X stock concentration. Your final reaction must contain 1X PCR mix. How much of the 2X PCR mix will you need to add? c) We will use 1 L of the genomic DNA isolated from Arabidopsis as our template for PCR. Fill in the table. Have the instructor check your numbers before you set up your reaction. Component | amount in L 2X PCR mix | 5 M primer 1 | 5 M primer 2 | genomic template DNA | 1 water | (total volume) | 25
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Complete this master mix table for 5 DNA samples. When deciding how many reactions to calculate, be sure to include the necessary controls required to interpret the PCR result relative to contamination and functional reagents and equipment, and be sure to account for pipetting error. Show your work (attach extra sheet if necessary). (5 points) Draw out your pipetting scheme for your Master Mix and your PCR reactions. Draw a single 1.5 ml Eppendorf tube for your Master Mix (MM) and 0.2 ml PCR tubes for each of your PCR reactions. Label your drawings, indicating what each tube contains (e.g. "39 ul of MM and 1 ul of DNA"). (4 points)
The PCR reactions consist of primers, cell suspension; and PCR Master-Mix at a final volume of 50 pl: The Master-Mix contains PCR buffer; dNTPs, Taq polymerase and water: Each of the 50 ul reactions requires 1X PCR buffer; 0.2 mM dNTPs, and 1.25 Units of Taq polymerase_ Given the following stock concentrations: 10X PCR buffer; 10 mM dNTPs, and 5 Units/ul Taq polymerase, determine how you would prepare just enough of the Master-Mix for the six PCR reactions performed in your experiment: Water is used as the solvent in preparing the Master-Mix: Note: The final volume (50 pl) of each PCR reaction contains primers 4l) , extracted DNA (4 pl); and the master mix 2_ Why do we plug the black cable in at the top of the gel and the red cable at the bottom? What property of the DNA are we using during electrophoresis? 3_ Using _ the ''pseudomonad-specific"' primers, does obtaining band of approximately 1300 bp definitively establish your isolate O unknown is a pseudomonad? Does your negative control lane produce any bands (e.g , ~false positive" )? Are the bands produced by the isolates and unknowns the same size? What might account for the difference in size?
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