18. You are preparing a PCR for 8 samples, including the negative control, for 25µL reactions. Use the table below to calculate your volumes to make the master mix of your reaction. Make sure to buffer for an extra sample or two in case you mess up. (27 points) # of samples: 8 Ingredient PCR tube volume MM volume dH20 2.5µL Forward primer 3µL Reverse primer 3µL EconoTaq PLUS 2X 12.5µL Total Volume: How much master mix is added to each tube? How much DNA is added to each tube?
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The total volume of the PCR reaction is the sum of the volumes of all the ingredients added to each tube. In this case, the total volume is 25 μL. Show more…
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You have been assigned the task of performing PCR on seven different experimental DNA samples, and need to prepare a master mix prior to setting up the PCR reaction. Listed below are the reagents and volumes needed for a single reaction. Some of the volumes are missing from the table. Using the information available in the table and what you know about setting up PCR reactions, complete the table below. Reagent | One Reaction | Master Mix (7 Rxns) Water | | 10X Reaction Buffer | | Forward Primer | 1.0 ul | Reverse Primer | | dNTP Mix 2.5mM each | | DNA polymerase | 0.5 ul | DNA | 1 ul | Final volume | 50 ul | When preparing the master mix, should the DNA template be added prior to aliquoting the reactions into separate PCR tubes? Explain your answer. No,
Adi S.
8. Complete this master mix table for 6 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) Master Mix* | Conc. of Stock Solution | Final Conc. | ̑L/Rxn (total per tube) | Number of Reactions | Total ̑l Needed for master mix PCR buffer (1) | 25X | 1X | | | dNTP mix (3) | 10 mM | 200 ̑M | | | MgCl2 (4) | 30 mM | 2.5 mM | | | Forward Primer (5) | 25 ̑M | 0.8 ̑M | | | Reverse Primer (5) | 25 ̑M | 0.8 ̑M | | | Taq polymerase (6) | 7 U/̑L | 0.09 U/̑L | | | DNA | | | 1 ̑L | | Water (2) | | | | | Total volume of the entire reaction (̑Ls) | | | 40 ̑L | | 9. Draw a pipetting scheme/diagram for all your PCR reactions/tubes and indicate what each vial contains (e.g. "39 ul of MM and 1 ul of DNA"). (4 points)
Sri K.
Write out the PCR reaction mix if you are using as stock reagents: 5X buffer, dNTPs 10mM, primer mix 10 µM, Taq polymerase 25X. You need a 25 µl final volume, final concentrations of buffer 1X, dNTPs 0.4 mM, primer mix at 0.4 µM, and Taq polymerase at 1X, and you will use 3 µl of DNA template. 2. What is happening at each stage of the PCR reaction? Step 1, 95°C, 3 minutes: Step 2, 95°C, 30 seconds: Step 3, 60°C, 30 seconds: Step 4, 72°C, 1 minute: Repeat of steps 2 – 4, 30 times: Step 5, 72°C, 3 minutes:
Jenny W.
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