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Plasmid preparation - Info

Plasmid preparation

Overview

 

What is a plasmid preparation?

 

In addition to the bacterial chromosome, bacteria possess so-called extrachromosomal plasmids on which genes are contained. A characteristic feature of plasmids is their ring shape. They are double-stranded DNA molecules that are able to replicate themselves. For this purpose, a plasmid has a sequence called the origin of replication (ORI).

In modern science, plasmids are used to duplicate genetic material within bacteria and to transfer foreign genes to other organisms.

The process by which plasmid DNA is isolated from transformed bacteria is called plasmid preparation.

Why do you do a plasmid preparation?

 

Such a plasmid preparation allows scientists to work with the plasmid DNA in vitro, i.e. outside the cell.

The entire process of plasmid preparation can be divided into two sub-steps. First, the cell content is released by the destruction of the cell walls. This step is also known as "unlocking" the cell. The cell extract obtained is then treated in such a way that all components except for the desired plasmid DNA are removed.

 

What types of plasmid preparation kits exist?

 

Plasmid preparation kits provide the scientist with all necessary tools to perform such a plasmid preparation. Based on the amount of plasmid DNA that can be isolated, these kits are divided into four groups.

A miniprep kit allows the isolation of up to 25 µg plasmid DNA.

Midiprep kits are used for quantities up to 0.5 mg.

Larger amounts up to 2 mg are isolated using a maxiprep kit.

If these plasmid DNA amounts are not sufficient, a gigaprep kit can be used, which can isolate up to 2 mg plasmid DNA.

 

A selection of our plasmid preparation kits

 

Product Manufacturer Item No. Amount
QuickClean II Plasmid Miniprep Kit

GenScript

L00420-50

50 rxns

Plasmid DNA Extraction Mini Kit (100prep)

Favorgen

FAPDE100
FAPDE001

100 preps
100 preps

QuickPure Plasmid Mini kit

CoWin Biosciences

FAPDE100

200 rxns

EZ-10 Spin Column Endotoxin-Free Plasmid DNA Mini-Preps Kit

Bio Basic

BB-BS71918

50 preps

FavorFilter Plasmid Midi SP Kit (25prep)-­spin column

Favorgen

FAPDE005-M-025

25 preps

GoldHi EndoFree Plasmid Midi Kit

CoWin Biosciences

CWB-CW2581S

10 rxns

Plasmid DNA Maxi Endotoxin Removal kit(10prep)-spin column

Favorgen

FAPDE005C-010

10 preps

GoldHi EndoFree Plasmid Maxi Kit

CoWin Biosciences

CWB-CW2104M

10 rxns

EZ-500 Spin Column Endotoxin-Free Plasmid DNA Maxi-Preps Kit

Bio Basic

BB-SK1250

20 preps

 

What are the differences in a plasmid preparation?

 

The plasmid preparation can be carried out by means of alkaline lysis or Koch lysis. As the name suggests, the two procedures differ in the methodology for lysis of the bacterial cell wall.

In alkaline lysis, a detergent called sodium dodecyl sulfate (SDS) is used to dissolve the bacterial cell wall and thus make the contents accessible. The addition of sodium hydroxide then causes the pH to rise to alkaline. This has the consequence that the hydrogen bridge bonds between the DNA strands are released. In contrast to chromosomal DNA, however, plasmid DNA is capable of being completely renatured afterwards. The remaining components are then simply centrifuged so that the plasmid DNA remains in the supernatant.

In the case of cooking lysis, the bacterial cell wall is dissolved with the help of the enzyme lysozyme. This is followed by denaturation of the bacterial proteins with isopropyl alcohol. In this procedure, the chromosomal DNA and denatured protein are also centrifuged and the plasmid DNA is extracted from the supernatant.