Answered: D. PCR of lacZ GENE 1. You will be…

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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If you wanted to prepare only one PCR reaction, how much of each reagent would you add to the PCR tube?

Transcribed Image Text:D. PCR of lacz GENE 1. You will be preparing a PCR reaction for your "G" and "L" tubes. When performing a PCR it is important to make sure the reaction has worked correctly, therefore we will also be preparing a "positive control". In addition, we want to make sure that there has not been any contamination so we will prepare a "negative control". We will first prepare a "MASTER-MASTER MIX" for all the reactions. 2. Obtain a sterile 1.5-ml tube. 3. Add the following reagents to the tube to make the "MASTER-MASTER MIX": Reagent GoTag® Green Master Mix lacZ forward primer (10 µM) lacZ reverse primer (10 µM) Nuclease free water Volume 125 ul 12.5 ul 12.5 µl 90 ul 4. Obtain 4 PCR tubes and a PCR tube rack. Label the tubes: G, L, + and -. 5. Obtain your "G" and "L" CDNA samples from last week and a + PCR control. Place on ice. 6. Add 48 ylof the mix to each PCR tube. (NOTE: You will have left over mix because we prepared enough mix for 5 reactions.) 7. Add 2 µl of nuclease free water to the tube labeled -. 8. Add 2 µl of "G" CDNA to the PCR tube labeled "G". 9. Add 2 ul of “L" CDNA to the PCR tube labeled "L". 10. Add 2 µl of positive control to the tube labeled +. 11. Place tubes in the thermocycler. 12. The thermocycler profile is as follows: o 94°C for 10 min o 35 cycles of: 94°C for 30 sec • 60°C for 60 sec 72°C for 60 sec o 72°C for 10 min

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