Question

5. You successfully amplify the above Gene A sequence and plan to clone the PCR fragment into restriction enzyme sites shown in the figure of plasmid 1. The promoter in the plasmid should result in transcription of Gene A if the molecular cloning is successful. The arrow indicates start & direction of transcription. The resulting recombinant plasmid (Plasmid1-GeneA) would express Gene A in E.coli cells after transformation. Stul recognition site 5'...AGGCCT...3' 3'...TCCGGA...5' Sall recognition site 5'...GTCGAC...3' 3'...CAGCTG...5' Promoter Stul Sall EcoRI Plasmid 1 EcoRI recognition site 5'...GAATTC...3' 3'...CTTAAG...5' Given the DNA sequence of Gene A (previous page), plasmid map and restriction enzyme recognition sites, there are two ways to insert amplified Gene A sequence into plasmid 1. a. Look for the above restriction enzyme sites in DNA sequence of Gene A (previous page), can you cut this sequence using all of the above enzymes? Yes/No, which ones? (1 point) b. Which of the following options would you use to create the recombinant plasmid that would express Gene A in E.coli? Explain briefly why? (2 points) Option 1 Cut plasmid with | Cut Gene A with Option 1: Stu I and Sal I | Stu I and Sal I Option 2: Eco RI and Sal I | Eco RI and Sal I

          5. You successfully amplify the above Gene A sequence and plan to clone the PCR fragment into restriction enzyme sites shown in the figure of plasmid 1. The promoter in the plasmid should result in transcription of Gene A if the molecular cloning is successful. The arrow indicates start & direction of transcription. The resulting recombinant plasmid (Plasmid1-GeneA) would express Gene A in E.coli cells after transformation.

Stul recognition site
5'...AGGCCT...3'
3'...TCCGGA...5'

Sall recognition site
5'...GTCGAC...3'
3'...CAGCTG...5'

Promoter
Stul Sall
EcoRI
Plasmid 1

EcoRI recognition site
5'...GAATTC...3'
3'...CTTAAG...5'

Given the DNA sequence of Gene A (previous page), plasmid map and restriction enzyme recognition sites, there are two ways to insert amplified Gene A sequence into plasmid 1.

a. Look for the above restriction enzyme sites in DNA sequence of Gene A (previous page), can you cut this sequence using all of the above enzymes? Yes/No, which ones? (1 point)

b. Which of the following options would you use to create the recombinant plasmid that would express Gene A in E.coli? Explain briefly why? (2 points)
Option 1

Cut plasmid with | Cut Gene A with
Option 1: Stu I and Sal I | Stu I and Sal I
Option 2: Eco RI and Sal I | Eco RI and Sal I

5. You successfully amplify the above Gene A sequence and plan to clone the PCR fragment into restriction enzyme sites shown in the figure of plasmid 1. The promoter in the plasmid should result in transcription of Gene A if the molecular cloning is successful. The arrow indicates start direction of transcription. The resulting recombinant plasmid (Plasmid1-GeneA) would express Gene A in E.coli cells after transformation.

Stul recognition site
5'...AGGCCT...3'
3'...TCCGGA...5'

Sall recognition site
5'...GTCGAC...3'
3'...CAGCTG...5'

Promoter
Stul Sall
EcoRI
Plasmid 1

EcoRI recognition site
5'...GAATTC...3'
3'...CTTAAG...5'

Given the DNA sequence of Gene A (previous page), plasmid map and restriction enzyme recognition sites, there are two ways to insert amplified Gene A sequence into plasmid 1.

a. Look for the above restriction enzyme sites in DNA sequence of Gene A (previous page), can you cut this sequence using all of the above enzymes? Yes/No, which ones? (1 point)

b. Which of the following options would you use to create the recombinant plasmid that would express Gene A in E.coli? Explain briefly why? (2 points)
Option 1

Cut plasmid with | Cut Gene A with
Option 1: Stu I and Sal I | Stu I and Sal I
Option 2: Eco RI and Sal I | Eco RI and Sal I

Added by Jose B.

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