The table below shows restriction enzyme pairs used by various teams to digest the YFG and clone it in the bacterial plasmid cut with the same enzymes. Complete the table below for each restriction enzyme pair.
As a part of undergrad lab project, you have to clone Your Favorite Gene into plasmid cloning vector (4740 bp) pUC18. Restriction maps for both YFG and cloning vector are provided.
The plasmid has a bacterial origin of replication (ori), an ampicillin resistance gene (AmpR) that degrades ampicillin antibiotic. It also has LacZ gene that encodes the β-gal enzyme, which converts white X-gal substrate into a blue product. pLac is a bacterial promoter.
Restriction Enzyme Recognition Site : A slash (/) represents the cut site for each restriction enzyme.
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Enzyme 1 |
5’- G/TCGA C -3’ 3’- C AGCT/G-5’ |
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Enzyme 2 |
5’-C/TCGA G-3’ 3’G AGCT/ C5’ |
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Enzyme 3 |
5’- GTC / GAC- 3’ 3’- CAG / CTG- 5’ |
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Enzyme 4 |
5’-C AATT /G-3’ 3’-G/ TTAA C-5’ |
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Enzyme 5 |
5’C AATT/G-3’ 3’G/TTAA C-5’ |
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Enzyme 6 |
5’G/GATC C-3’ 3’C CTAG/ G-5’
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Enzyme 7 |
5’G/GATC C-3’ 3’C CTAG/ G-5’ |
1a)
The table below shows restriction enzyme pairs used by various teams to digest the YFG and clone it in the bacterial plasmid cut with the same enzymes.
Complete the table below for each restriction enzyme pair.
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Restriction enzymes used to cut YFG |
Will you be able to… |
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Column 1: Clone the digested YFG into the plasmid –
YES/NO? |
Column 2: Amplify the recombinant plasmid in the ampicillin sensitive bacterial cells: YES/NO? |
Column 3: Express YFG in the bacterial cells:
YES/NO/Maybe? |
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1 and 5 |
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1 and 2 |
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2 and 6 |
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3 and 7 |
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