Human Anatomy & Physiology (11th Edition)
Human Anatomy & Physiology (11th Edition)
11th Edition
ISBN: 9780134580999
Author: Elaine N. Marieb, Katja N. Hoehn
Publisher: PEARSON
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Genome annotation
The technique of linking biological information to genome sequences is termed genome annotation. Gene annotation is the method of identifying gene locations and coding sections. It helps us understand what these genes are doing in the body through establ…
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  1. What is the final volume of the individual PCR reactions we are making?       
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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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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