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Introduction

Plasmids are
extrachromosomal DNA that naturally present in prokaryotes cells. The
characteristics of plasmids are it can self-replicating, stable and easy to
isolate and manipulate due to its smaller size. These features enable
researchers to use it as a tool for genetics study and in recombinant DNA
technology.  Usually plasmids contain
origin of replication (ORI), antibiotic resistance gene and multiple cloning
site. Multiple cloning site is essential part of the plasmids for DNA
recombination, where it contains at least one restriction site to enable the
insertion of the desired DNA fragment by the restriction enzyme. These plasmid
vector or recombinant plasmid are then inserted into bacterial cells by transformation
and the desired DNA fragment can then be produced in huge amounts.

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Objective

To extract and
purify wild type plasmid (sf1044) and own plasmid (pNV1685).

 

Materials and Apparatus

Cell resuspension
solution, cell lysis solution, neutralization solution, column wash solution,
minicolumns, collection tube, alkaline protease solution, nuclease-free water,
microcentrifuge tube, microcentrifuges, pipette and tips.

 

Procedures

As lab manual and
Wizard Plus Minipreps DNA Purification System Protocol

 

Result and Discussion

The plasmid
extraction and purification consists of several important steps. First, the
cells are resuspended in TE buffer which contains Tris and EDTA. This buffer
solution will cause cell walls to weaken and the DNases activities are
prevented. Second, the cell is lysed by using cell lysis solution that contains
NaOH and SDS, which causes the cell wall to break down and denature the dsDNA
to ssDNA. Third, the plasmid ssDNA will convert back to dsDNA when treated with
potassium acetate in the neutralization solution. Centrifugation step will
separate the gDNA and denatured cellular protein from the plasmid DNA. Lastly,
plasmid DNA is precipitated by using ethanol, centrifuged and stored at –20°C
for subsequent use (G-Biosciences, 2013)

 

According to the
Thermo Scientific Technical Bulletin for NanoDrop Spectrophotometer, the
concentration and the purity of the extracted plasmid are checked with Thermo
Scientific NanoDrop Spectrophotometer, where it measures the absorbance of all
molecules in the sample. The purity of the plasmid DNA is determined with the
260/280 absorbance ratio and the ratio reading of approximately 1.8 and above
indicates the “purity” of the DNA samples. Thus, purification steps of the
plasmid are essential to ensure the readings taken are accurate.

 

In this practical,
we extracted and purified wild type plasmid sf1044 and own plasmid pNV 1685 with
concentration of 513.6 ng/µl and 370.8 ng/µl respectively. Both plasmids also
yielded more than 2.0 purity of 260/280 absorbance ratio (Table 1).

 

 Table
1: The concentration and purity of the extracted wild type plasmid and own
plasmid.

 

Concentration (ng/µl)

Purity (260/280)

Wild type plasmid (sf1044)

513.6

2.11

Own plasmid (pNV1685)

370.8

2.09

 

The wild type plasmid sf1044 provided in
this practical is a high copy number plasmid compared to plasmid pNV1685. Thus,
it yielded higher concentration when extracted and purified. Plasmid with
higher copy number is usually more preferred for cloning and protein expression
since it can produce a higher yields and results (Karen, 2016).  The extracted wild type plasmid was selected
and stored for the next practical.

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