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Note: the reaction volumes will be 20 μL and n = 10 reactions! Assume you extract gDNA quantitated by the NanoDrop at a concentration of 1065.7 ng/μL. Calculate the dilution(s) needed to get to a concentration of 25 ng/μL needed for making your Master Mix. Show pipettable volumes! Do two-step dilution.... Show work.

Note: the reaction volumes will be 20 μL and n = 10 reactions! Assume you extract gDNA quantitated by the NanoDrop at a concentration of 1065.7 ng/μL. Calculate the dilution(s) needed to get to a concentration of 25 ng/μL needed for making your Master Mix. Show pipettable volumes! Do two-step dilution.... Show work.

Biochemistry
Biochemistry
9th Edition
ISBN:9781319114671
Author:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Publisher:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Chapter1: Biochemistry: An Evolving Science
Section: Chapter Questions
Problem 1P
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### Making Your Master Mix and PCR Setup for TPCN2 #### Note: The reaction volumes will be 20 µL, and \( n = 10 \) reactions. --- **1. DNA Dilution Instructions:** Assume you extract genomic DNA (gDNA) quantified by the NanoDrop with a concentration of 1065.7 ng/µL. Calculate the dilution(s) necessary to achieve a target concentration of 25 ng/µL for preparing your Master Mix. Use a two-step dilution method and clearly show the pipettable volumes alongside your calculations. **2. dNTPs Dilution Instructions:** Your deoxynucleotide triphosphates (dNTPs) are provided separately in 100 mM concentrations for ATP, GTP, CTP, and TTP. Indicate the mixture components and volumes. Calculate the necessary dilution(s) to prepare a dNTP mixture at 2.5 mM for the Master Mix. Ensure there is an adequate amount for all your reactions. **3. Primer Preparation Instructions:** Your Forward and Reverse Primers for TPCN2 are re-suspended at 100 µM. Calculate the dilution(s) required to prepare the primers in such a way that they can be pipetted appropriately for creating your Master Mix. Use the specified concentrations from the setup sheet. --- The details above will guide you through the process of preparing your PCR reactions efficiently, ensuring all components are accurately quantified and diluted for optimal results.
Transcribed Image Text:### Making Your Master Mix and PCR Setup for TPCN2 #### Note: The reaction volumes will be 20 µL, and \( n = 10 \) reactions. --- **1. DNA Dilution Instructions:** Assume you extract genomic DNA (gDNA) quantified by the NanoDrop with a concentration of 1065.7 ng/µL. Calculate the dilution(s) necessary to achieve a target concentration of 25 ng/µL for preparing your Master Mix. Use a two-step dilution method and clearly show the pipettable volumes alongside your calculations. **2. dNTPs Dilution Instructions:** Your deoxynucleotide triphosphates (dNTPs) are provided separately in 100 mM concentrations for ATP, GTP, CTP, and TTP. Indicate the mixture components and volumes. Calculate the necessary dilution(s) to prepare a dNTP mixture at 2.5 mM for the Master Mix. Ensure there is an adequate amount for all your reactions. **3. Primer Preparation Instructions:** Your Forward and Reverse Primers for TPCN2 are re-suspended at 100 µM. Calculate the dilution(s) required to prepare the primers in such a way that they can be pipetted appropriately for creating your Master Mix. Use the specified concentrations from the setup sheet. --- The details above will guide you through the process of preparing your PCR reactions efficiently, ensuring all components are accurately quantified and diluted for optimal results.
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