Insertion and amplification of EGFP protein into pET41a(+) Plasmid
Insertion and amplification of EGFP protein into pET41a(+) Plasmid
Introduction
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
Aqueorea victoria jellyfish, GFP , when shown under UV light produces a green (flourescent)
color allowing the protein to be easily noticeable in most living organisms.(1) This works out
best for gene expression using the pET41a(+) vector plasmid. For this experiment, ligation is
used to insert the gene into the pET-41a(+) vector, then transforming it into E.coli to replicate
and create clones of e.coli producing large numbers of recombinant expression plasmid. The
plasmid DNA miniprep is then used to isolate the cloned EGFP expression plasmids. Restriction
digests and the PCR experiment help analyze these test and determine if a new recombinant
DNA plasmid has formed.
The ligation process of the experiment and the goal of which to successfully ligate EGFP into
the the prepared pET-41 plasmid takes place in the following steps. The outcome that is desired
is the creation of recombinant pET-41/EGFP plasmid. To forward this reaction, two concentra- tions of ligations were used. The first being 25 ng/μl cut pET41a and adding 2μl of this to obtain
50 ng NcoI/NotI cut pET-41a and inserting to 1μl 7 ng EGFP 1:1 molar ratio, and the second 1μl
of 50 ng NcoI/NotI pET-41a to 21 ng EGFP
Introduction
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
Aqueorea victoria jellyfish, GFP , when shown under UV light produces a green (flourescent)
color allowing the protein to be easily noticeable in most living organisms.(1) This works out
best for gene expression using the pET41a(+) vector plasmid. For this experiment, ligation is
used to insert the gene into the pET-41a(+) vector, then transforming it into E.coli to replicate
and create clones of e.coli producing large numbers of recombinant expression plasmid. The
plasmid DNA miniprep is then used to isolate the cloned EGFP expression plasmids. Restriction
digests and the PCR experiment help analyze these test and determine if a new recombinant
DNA plasmid has formed.
The ligation process of the experiment and the goal of which to successfully ligate EGFP into
the the prepared pET-41 plasmid takes place in the following steps. The outcome that is desired
is the creation of recombinant pET-41/EGFP plasmid. To forward this reaction, two concentra- tions of ligations were used. The first being 25 ng/μl cut pET41a and adding 2μl of this to obtain
50 ng NcoI/NotI cut pET-41a and inserting to 1μl 7 ng EGFP 1:1 molar ratio, and the second 1μl
of 50 ng NcoI/NotI pET-41a to 21 ng EGFP