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Plasmid Mapping Activity Answers

The document outlines the steps for transferring a useful gene from bacteria to a plant, including isolation, fragmentation, and amplification of the DNA. It details the use of restriction enzymes, gel electrophoresis, and Polymerase Chain Reaction (PCR) to prepare the gene for insertion. Finally, it describes the transformation of Agrobacterium to facilitate the incorporation of the gene into the plant genome.

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Mark Angelo Austria
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23 views2 pages

Plasmid Mapping Activity Answers

The document outlines the steps for transferring a useful gene from bacteria to a plant, including isolation, fragmentation, and amplification of the DNA. It details the use of restriction enzymes, gel electrophoresis, and Polymerase Chain Reaction (PCR) to prepare the gene for insertion. Finally, it describes the transformation of Agrobacterium to facilitate the incorporation of the gene into the plant genome.

Uploaded by

Mark Angelo Austria
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as DOCX, PDF, TXT or read online on Scribd

Activity1: Genetic Engineering

Name: ___________________________ Section:


_____________

Note: No cellphone allowed during the activity. No cellphone allowed.

Instruction: You have identified a useful gene in bacteria. Make a flow chart of
the steps that you would follow to transfer this gene to a plant. Draw this on one
whole sheet (white) paper.

Isolation of ____(1)____

Fragmentation of DNA by ____(2)_____

Separation and Isolation of desired ______(3)_______


____________

_______(4)_______ of gene of interest using _(5)_

_______(6)_______ of the DNA fragment into the vector

Transferring of the recombinant DNA into the __(7)__.

Solution:
1. The bacteria which contain the gene of interest is grown and harvested.
The bacterial cells were digested by lysozyme enzymes.
The DNA is concentrated by appropriate treatments and the purified DNA is
precipitated by adding chilled ethanol. The Ti plasmid (vector) is also isolated
from Agrobacterium.

2. Digestion of both the DNA and the vector (Ti plasmid) is performed by
incubating the purified DNA and vector separately with restriction enzymes.
Both the vector and DNA with the gene of interest are digested with the same
restriction enzyme.

3. After restriction enzyme digestion, separation of DNA fragments is carried


out by gel electrophoresis. The fragments are separated based on their size.
Separated bands of DNA on the gel plate, are extracted by elution. (Elution is
the process of extracting one material from another by washing with a
solvent.)

4. -5. After elution the DNA fragments are subjected to Polymerase Chain
Reaction (PCR).
The desired gene of interest is amplified to millions of copies. It takes place in
three steps like denaturation, annealing and extension. The primer, Taq
polymerase and deoxynucleotides are used for this procedure.

6. The amplified gene is ligated with the Ti plasmid in the T - DNA region. Thus,
the gene of interest inactivates the tumor inducing capability of Ti plasmid.
7. The Ti plasmid is injected into the Agrobacterium host by transformation.
Then, the Agrobacterium is made to infect the host (plant cell) and the gene
of interest present in the T-DNA region is incorporated into the plant genome.

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