Extration principle,extraction operation and Enzymatic Digestion of Plasmid DNA在线视频

Plasmids are small circular molecules of double stranded extrachromosomal DNA

found in bacteria and some other organisms.

They are important tools in genetic and biotechnology labs.

They are used in recombinant DNA experiments

to clone genes from other organisms

and make large quantities of their DNA.

The minipreparation of plasmid DNA

is a small-scale isolation of plasmid DNA from bacteria.

Restriction endonucleases are enzymes

that make double stranded cuts in DNA molecules.

These enzymes recognize specific DNA sequence

and cleave each DNA strand.

Agrose gel electrophoresis is a powerful separation method

frequently used to analyze plasmid DNA.

Bacteria contain one or more plasmids in it

which present in the form of a number of copies in each cell. 

The plasmid DNA molecule is smaller than the chromosomal DNA,

and has a supercoil closed ring structure. 

This method is based on the different rates of denaturation

and renaturation of covalently closed circular plasmid DNA

and chromosomal DNA.

The main component of Resuspension Buffer are glucose,

Tris-HCl hydrochloric acid，EDTA and RNase.

RB solution resuspend the bacteria precipitate.

The main component of Lysis buffer

are sodium hydroxide(NaOH) and SDS.

Bacteria were lysed with alkaline lysis buffer consisting of SDS.

SDS is a detergent, cleaves the phospholipid bilayer of membrane.

Sodium hydroxide (NaOH) denatures both the chromosomal

and plasmid DNA into single strands,

and it also denatures the proteins

which are involved in maintaining the structure of the cell membrane.

The main component of Neutralization Buffer

are Acetate and Potassium acetate.

Acetate and potassium acetate

will neutralize the PH and allow DNA to be renatured.

However, the large chromosomal strands cannot rehybridize perfectly,

potassium acetate also precipitate SDS

with lipids and protiens from the solution.

The associated SDS lipids protein traps the tangled chromosomal DNA

in the precipitate

Only the plasmid DNA,

small fragments of chromosomal DNA,

and RNA remain in the solution. 

So, through a series of steps involving

agitation, precipitation, centrifugation

and removal of supernatant,

the plasmid gets isolated and purified

Digestion of plasmid DNA with restriction endonuclease (RE)

RE can make double stranded cuts in DNA molecules.

The blue part in the circular DNA is plasmid DNA,

the red part is the inserted target DNA.  

The recognition sequence of RE is called palindromic structure,

also called inverted repeat sequence 

Bacteria

EasyPure Plasmid MiniPrep Kit,

the kit includes,

Resuspension Buffer(RB)

Lysis Buffer(LB, Blue)

Neutralization Buffer(NB,Yellow)

Wash Buffer(WB)

Elution Buffer( EB)

Mini-plasmid Spin Columns with Collection Tubes

DNA marker

Loading Buffer

Restriction endonuclease:

XbaⅠ

Hind Ⅲ

Agarose

Eppendorf tube

Pipettor

Centrifuge

Electrophoresis System

Water bath 

Step 1: Collect the bacteria

1ml bacteria solution are transferred to the eppendorf tube,

Centrifuge at 10000 g for 1min,

And then discard supernatant.

Collect the bacteria precipitate.

Step 2

Resuspend the bacteria.

Resuspend the bacteria precipitate in 250 μl of Resuspension Buffer (RB) .

Step 3: Lysis the bacteria.

Add 250 μl of Lysis Buffer (LB)

and mix well by inverting gently 5 times.

Make the solution change to transparent blue,

this step should not exceed 5 minutes. 

Step4

Add 350 μl of Neutralization Buffer (NB)

and mix very gently 5 times .

The color will change from blue to yellow,

and form yellow agglutinator.

Stand at room temperature for 2 minutes

Centrifuge at 12000 g for 5 minutes. 

Step 5

Transfer 800μl supernatant into the Spin Column.

Centrifuge at 12000 g for 1 minute.

The effluent in the Collection Tubes was discarded.

Place the Spin column in the collection tubes again.

Step 6

Add 650 μl of Wash Buffer ( WB )

and centrifuge at 12000 g for 1 minute.

Discard the effluent.

Put the column back into the Collection Tubes

Step 7

Centrifuge at 12000 g for 2 minutes.

Thoroughly remove the residual Wash Buffer

Step 8

Remove the column in a new Eppendorf tube.

Wait 5-10 minutes for ethanol to evaporate thoroughly

(The Elution Buffer is preheated in 60-70 degree water bath)

Add 30μl of Elution Buffe at the center of the column.

Stand at room temperature for 1 minute.

Centrifuge at 12000g for 1 min,

the effluent is plasmid DNA.

Step 9

Enzymatic Digestion

Digestion of plasmid DNA with restriction endonuclease.

The enzyme digestion system was constructed

Pipet the following solutions into a microcentrifuge tube.

Plasmid DNA: 10μl

Deionized water: 6μl

10×Reaction Buffer: 2μl

Hind Ⅲ: 1μl

XbaⅠ: 1μl

Tab the tube to mix the contents

Incubate at 37℃ water bath for 2 hours. 

Step10

The results were detected by agarose gel electrophoresis.

Prepare 1% agarose gel.

Place the gel onto the electrophoresis tank.

Fill the tank with proper amount of TAE buffer.

Add 5ul of loading buffer

to the plasmid DNA or the enzymatic production.

Load 10ul of this mixture to the agarose gel well

Connect the gel tank with the power supply properly.

Allow electrophoresis to progress for appropriate time.

Observe the results under the ultraviolet.

This picture show the results.

M refer to DNA Marker

Line 1,2,5,6,7,8 were the enzymatic digestion results,

there are two band,

refer to the target DNA and linear plasmid DNA respectively.

Line 3,4 were the plasmid isolation results,

only one band which refer to the super coiled plasmid DNA.