Insertion and amplification of EGFP protein into pET41a(+) Plasmid

3727 Words Jul 13th, 2014 15 Pages
Insertion and amplification of EGFP protein into pET41a(+) Plasmid


The overall purpose of the experiments conducted is to test the creation of recombinant

plasmid using recombinant DNA technology. The research of recombinant DNA began with the

use of E.Coli (Escherichia coli), a common bacteria found in the intestines of warm-blooded or- ganisms (5). Scientists worked together and generated a way, from cloning and using recombi- nant research, to achieve recombinant DNA. The gene that is the focus of this experiment and is

used for copying and expressing is EGFP. EGFP, which is the enhanced version of GFP (Green

Fluorescent Protein) upon mutation, is perfect for the expression of genes. Cloned from the
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After mixing and acquiring correct volume amount for

digest, incubate at 37° C for 1 hour. Each plasmid will also have an undigested sample, which

will have the same reagents excluding the added enzyme. These samples are ran on a 50 ml .9%

agarose gel at 110 volts with 4μl 6X track dye added for about 40 minutes.

Polymerase Chain Reaction (PCR)

The goal of the PCR experiment was to identify the recombinant plasmids. This will help to

confirm that the right fragment of gene was amplified according to the size and amount. The iso- lated plasmids from earlier are used in the the PCR experiment. Two primers are used for this,

pAD1sense (specific for EGFP insert) and pAD1anti (specific for pET-41 vector), along with a

positive control pET-41/EGFP, and a negative control (water). This negative control will help

determine the accuracy of the bands, and test for contamination. The reagents used for this reac- tion include Nuclease-free water, 2 μl of 10x PCR buffer, 1.6 μl of 10 mM dNTPS, 1 μl of

both10 μlM pAD1sense primer and 10 μlM pAD1anti primer, 0.5 μl of 5Units taq polymerase,

all with 2 μl of the plasmid DNA. Each PCR having a final total

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