Isolating Plasmid DNA

Summary

This video script offers a detailed guide on manipulating plasmids, circular DNA segments used in genetic engineering. It explains the process of introducing synthetic plasmids into bacteria, using them as vectors for gene replication and expression. The script outlines the steps for plasmid DNA extraction from bacteria, including transformation, isolation using a mini prep kit, and purification through centrifugation and silica matrix binding. The final product is purified plasmid DNA, ready for use in various biological applications.

Takeaways

• 🧬 A plasmid is a circular, non-chromosomal DNA segment found in some bacteria and is used as a vector for genetic engineering.
• 🔬 Biologists can manipulate plasmids in vitro and introduce them into bacteria, often using selectable markers like antibiotic resistance genes.
• 🔄 Plasmids typically include an origin of replication, a multiple cloning site, and the gene or DNA sequence of interest.
• 🌱 Transformation is the process used to introduce plasmids into bacteria, which is not always 100% efficient but can be made efficient through antibiotic selection.
• 📈 Bacterial cells can replicate plasmid DNA, allowing for the production of large amounts of specific DNA sequences.
• 🧪 Plasmid isolation kits, or minir prep kits, are used to extract plasmid DNA from bacteria through a series of steps involving buffers and a purification column.
• 🧫 The process begins with an overnight culture of transformed bacteria, followed by centrifugation to separate bacterial cells from the growth medium.
• 🌡️ Resuspension and lysis buffers are used to break open the cells and release plasmids, with care taken to avoid shearing genomic DNA.
• 🌀 Neutralization buffer is added to precipitate cellular debris and leave plasmid DNA in solution, followed by centrifugation to separate the components.
• 🧴 The supernatant containing plasmid DNA is passed through a spin column with a silica matrix that binds to the DNA, allowing for purification.
• 💧 Finally, elution buffer is used to release the purified plasmid DNA from the silica matrix, yielding the desired DNA for further use.

Q & A

What is a plasmid?

A plasmid is a circular, non-chromosomal segment of DNA that exists in some bacteria and can be manipulated by biologists for various genetic engineering purposes.

How are plasmids used in genetic engineering?

Plasmids are used as vectors to replicate or express particular genes or DNA sequences of interest that have been inserted into them.

What is a selectable marker on a plasmid?

A selectable marker, such as an antibiotic resistance gene, is a feature on a plasmid that allows for the selection of cells that have successfully taken up the plasmid.

What is the purpose of an origin of replication on a plasmid?

The origin of replication is a specific DNA sequence that allows the plasmid to be replicated within a host cell.

What is a multiple cloning site or polylinker?

A multiple cloning site or polylinker is an area on a plasmid with many known restriction enzyme sites where a gene or DNA sequence of interest can be inserted.

How are plasmids introduced into bacteria?

Plasmids are introduced into bacteria through a process called transformation, which is not 100% efficient but can be made more successful with antibiotic selection.

Why is antibiotic selection used after introducing plasmids into bacteria?

Antibiotic selection is used to efficiently select for cells carrying the plasmid of interest because bacteria with the plasmid will be resistant to the antibiotic.

How can bacterial cells serve as factories for making large amounts of plasmid DNA?

Bacterial cells can serve as factories because they replicate plasmid DNA before each cell division, and some plasmids are present in multiple copies per cell, allowing for rapid production of plasmid DNA.

What is a plasmid isolation kit or mini-prep kit?

A plasmid isolation kit, sometimes called a mini-prep kit, contains various buffers and a column that binds to plasmid DNA to purify it from bacterial cells.

What are the steps involved in extracting plasmid DNA from bacteria using a mini-prep kit?

The steps include labeling tubes, harvesting bacterial cells by centrifugation, resuspending cells in resuspension buffer, lysing cells with L buffer, neutralizing with neutralization buffer, centrifuging to precipitate debris, passing the supernatant through a spin column, washing the column, and eluting the purified plasmid DNA with elution buffer.

Why is it important to balance the centrifuge when spinning bacterial cultures?

Balancing the centrifuge ensures even distribution of weight and prevents uneven spinning, which could affect the pellet formation and the quality of the plasmid DNA isolation.

How does the addition of neutralization buffer affect the DNA during the plasmid DNA extraction process?

The neutralization buffer returns the pH to neutral, allowing DNA strands to renature. Chromosomal DNA forms an insoluble precipitate with cellular debris, while plasmid DNA remains in solution.

What is the role of the silica matrix in the spin column during plasmid DNA purification?

The silica matrix in the spin column binds to the plasmid DNA, allowing it to be separated from other cellular components like RNA, proteins, and salts that pass through the membrane.

Outlines

00:00

🧬 Introduction to Plasmid Biology and Manipulation

This paragraph introduces plasmids as circular, non-chromosomal DNA segments found in bacteria, which can be manipulated by biologists. Plasmids are used as vectors to replicate or express specific genes or DNA sequences of interest. They typically contain a selectable marker, an origin of replication, a multiple cloning site, and the gene or DNA sequence of interest. The process of introducing plasmids into bacteria is called transformation, which is not 100% efficient but can be made efficient through antibiotic selection. Plasmids are used to produce large amounts of DNA molecules, and the process of extracting plasmid DNA from bacteria involves using a plasmid isolation kit, which includes various buffers and a column to purify the DNA. The initial steps of this protocol involve labeling tubes, pipetting cultures, and centrifugation to harvest bacterial cells.

05:00

🔬 Plasmid DNA Extraction Process

The second paragraph delves into the specifics of the plasmid DNA extraction process. After harvesting bacterial cells, the cells are resuspended in a buffer to release plasmids. An L buffer is added to lyse the cells and release their contents, including plasmid DNA. The solution becomes viscous, and care is taken not to shear the genomic DNA. A neutralization buffer is then added to precipitate cellular debris and denature chromosomal DNA, while allowing plasmid DNA to remain in solution. The mixture is centrifuged to separate the precipitate from the supernatant containing the plasmid DNA. The supernatant is transferred to a spin column containing a silica matrix that binds to the plasmid DNA, leaving other contaminants behind. The column is washed to remove salts and contaminants, and finally, elution buffer is added to release the purified plasmid DNA from the silica matrix.

10:02

🧪 Conclusion of Plasmid DNA Isolation

The final paragraph concludes the process of plasmid DNA isolation. It describes the final steps of adding the elution buffer to the spin column to solubilize the DNA, followed by centrifugation to collect the purified plasmid DNA in a microcentrifuge tube. The purified DNA is now ready for use in various molecular biology applications. The paragraph also instructs on discarding the used spin column and storing the tube containing the purified plasmid DNA, marking the successful completion of the plasmid DNA extraction protocol.

Keywords

💡Plasmid

A plasmid is a circular, non-chromosomal DNA molecule found in many bacteria and yeasts. In the context of the video, plasmids are manipulated by biologists to carry specific genes or DNA sequences of interest. They are used as vectors for genetic engineering, allowing for the replication and expression of inserted genes. The video describes how plasmids are introduced into bacteria and how they can be extracted for various applications, highlighting their importance in molecular biology.

💡Selectable marker

A selectable marker is a gene that provides a phenotype that can be selected for, often used in genetic engineering to identify cells that have successfully taken up a plasmid. In the video, an antibiotic resistance gene is mentioned as a common selectable marker, which allows for the selection of bacteria that contain the plasmid of interest due to their ability to survive in the presence of antibiotics.

💡Origin of replication

The origin of replication is a specific DNA sequence that serves as the starting point for DNA replication. In the script, it is mentioned as a component of plasmids that allows them to replicate within a host cell. This feature is crucial for the amplification of the plasmid and the genes it carries, making it possible to produce large quantities of the desired DNA sequences.

💡Multiple cloning site (polylinker)

A multiple cloning site, or polylinker, is a region within a plasmid that contains multiple recognition sites for restriction enzymes. This allows for the insertion of various DNA sequences of interest. The video emphasizes the importance of this feature in genetic engineering, as it facilitates the manipulation and cloning of specific genes into the plasmid.

💡Transformation

Transformation is a process by which cells take up foreign DNA, such as plasmids. In the video, it is described as the method by which synthetic plasmids are introduced into bacteria. The efficiency of this process is discussed, along with the use of antibiotic selection to ensure that only bacteria containing the desired plasmid are grown and selected.

💡Antibiotic selection

Antibiotic selection is a technique used to isolate bacteria that contain a plasmid with an antibiotic resistance gene. The video explains how this selection process is very efficient, as it allows for the identification and growth of bacteria that have successfully taken up the plasmid, which is crucial for genetic research and biotechnology applications.

💡Plasmid isolation kit

A plasmid isolation kit, also known as a mini-prep kit, is a set of reagents and materials used to extract plasmid DNA from bacterial cells. The video script describes the step-by-step process of using such a kit, which includes the use of various buffers and a column that binds to the plasmid DNA, ultimately purifying it from the bacterial cells.

💡Lysis buffer (L buffer)

Lysis buffer, or L buffer, is a solution used to break open bacterial cells and release their contents, including plasmid DNA. The script mentions the addition of L buffer to the resuspended bacterial cells, which helps in lysing the cells and preparing for the subsequent DNA extraction steps.

💡Neutralization buffer

Neutralization buffer is used to neutralize the alkaline conditions created by the lysis buffer, which helps in precipitating cellular debris while leaving the plasmid DNA in solution. The video describes the addition of this buffer after lysis, which is a critical step in the purification process to ensure the isolation of pure plasmid DNA.

💡Spin column

A spin column is a device used in molecular biology to separate and purify DNA based on its binding to a silica matrix within the column. The video script details the use of spin columns to bind and wash the plasmid DNA, removing contaminants before eluting the purified DNA with an elution buffer.

💡Elution buffer

Elution buffer is used to release the purified plasmid DNA from the silica matrix in the spin column. The video explains the final step of adding elution buffer to the spin column, allowing the DNA to be collected in a clean, purified form, ready for further use in molecular biology experiments.

Highlights

Plasmids are circular, non-chromosomal DNA segments found in bacteria.

Biologists can manipulate plasmids in vitro and introduce them into bacteria.

Plasmids used by geneticists often contain a selectable marker for identification.

Selectable markers may include antibiotic resistance genes.

Plasmids feature an origin of replication and a multiple cloning site.

The multiple cloning site has numerous known restriction enzyme sites.

Plasmids serve as vectors to replicate or express inserted genes or DNA sequences.

Transformation is the process used to introduce plasmids into bacteria.

Antibiotic selection is an efficient method for identifying bacteria with the desired plasmid.

Bacteria can replicate plasmid DNA, making them suitable for large-scale DNA production.

Plasmid isolation kits, or miniprep kits, are used to extract plasmid DNA from bacteria.

The isolation process begins with an overnight liquid culture of transformed bacteria.

Bacterial cells are separated from the liquid media by centrifugation.

Resuspension buffer is used to break open cells and release plasmids.

Lysis buffer (L buffer) is added to lyse cells and release plasmid DNA.

Neutralization buffer is used to precipitate cellular debris and leave plasmid DNA in solution.

Spin columns with silica matrices are used to bind and purify plasmid DNA.

Wash buffer is used to remove contaminants from the silica matrix.

Elution buffer is added to release purified plasmid DNA from the silica matrix.

The final solution contains isolated plasmid DNA ready for use or analysis.

Transcripts

a plasmid is a circular non-chromosomal

segment of DNA that exists in some

bacteria biologists can manipulate

plasmas in a test tube and introduce

these synthetic plasmids into

bacteria the plasmids geneticists

usually use have a selectable marker

such as an antibiotic resistance Gene An

Origin of replication a multiple cloning

site or polylinker that has many known

restriction enzyme sites and a gene or

DNA sequence of interest that is

inserted into the multiple cloning site

these artificial plasmids are used as

vectors to replicate or Express the

particular genes or DNA sequences of

interest that have been inserted into

the

plasmids plasmids are introduced into

bacteria through a process called

transformation although bacterial

transformation is far from 100%

efficient antibiotic selection for cells

carrying the plasmid of interest is very

efficient since bacteria replicate

plasma DNA before each cell division and

since some plasmids are present in

several copies per cell a simple

overnight culture of bacteria can yield

vast numbers of plasma DNA

molecules for this reason bacterial

cells can serve as factories for making

large amounts of plasma DNA and the DNA

sequence of

interest in order to extract plasma DNA

from bacteria

biologists use a plasmid isolation kit

sometimes called a plasmid minir prep

kit this kit consists of a variety of

buffers and a column that binds to the

plasma DNA to purify it biologists begin

with an overnight liquid culture of

transformed bacteria carrying the

desired plasma first they separate the

bacterial cells from the liquid media

then they resuspend the cells in a

resuspension buffer and lice or break

open the cells to release the plasmids

with a Lis buffer a neutralization

buffer is added to precipitate cellular

debris but leave the plasma DNA in

solution the tube is centrifuged to

separate the solid precipitate which

will be discarded from the liquid

supernatent the supernant containing the

plasmid DNA is passed through a spin

column containing a silica Matrix the

plasmid DNA binds to the silica Matrix

while the rest of the supernant passes

through the column and is discarded Ed

the column is then washed with wash

buffer to remove any remaining

contaminants finally an illusion buffer

is added to release the plasma DNA from

the silica Matrix this flow through is

caught in a fresh tube the final

solution contains isolated plasmid

DNA each step of this protocol will now

be demonstrated and explained in

detail label the 1.5ml microfuge tubes

you will need you will label one tube

per culture using sterile 5ml pipets

pipet 1.5 ml of each culture into the

appropriate labeled microfuge tube use a

fresh pipet for each culture to avoid

contamination you will now harvest the

bacterial cells from the culture by

centrifugation place the microfuge tubes

in a tabletop microcentrifuge and spin

for 1 minute at full speed be sure to

balance the centrifuge before starting

it this means that an identical tube

containing an identical volume of

solution should be directly across from

your tube don't forget to secure the

inner lid if your centrifuge requires

one at the end of the centrifugation a

small pellet will be at the bottom of

the tube this is made up of the

bacterial cells that contain the plasma

DNA you want the clear liquid

supernatent is lb the bacterial growth

media with an antibiotic such as

ampicillin remove all traces of growth

medium by pipetting or pouring out and

discarding the supernant don't worry the

pellet will not move as long as you

don't touch it in some cases you may

want to spin down an additional 1.5 ml

of bacterial culture in the same tube to

increase plasma DNA yield resuspend the

pelleted bacterial cells in 250 microl

of the resuspension buffer be sure to

use proper pipetting technique when you

do this after you have added the 250

microl of resuspension buffer mix until

no cell clumps are visible in the

suspension the resuspension buffer used

in this step contains rnas which will

degrade RNA from the minir prep in the

next step when the cells are lized once

all of the pellet in your tube has been

resuspended carefully add 250 microl of

the L buffer mix thoroughly by inverting

the tube 4 to 6 times the solution

should become viscous and slightly clear

do not allow the L to continue for more

than 5 minutes do not vortex at this

point or else the genomic DNA will be

sheared into small fragments and will

contaminate the plasma DNA if you were

using a kyogen kit the cell suspension

will turn blue after the addition of the

Lis buffer mix the solution until you

have a uniformly colored suspension the

Lis buffer contains a mixture of the

base sodium hydroxide and the detergent

sodium DL sulfate or SDS the SDS

dissociates the lip components of the

cell the cell membrane which is made up

of lipids breaks open or lices the

contents of the cell including the

cell's chromosomes and plasma DNA spill

out into the solution the SDS and high

pH of the solution then denature the

chromosomal and plasma

DNA working quickly add 350 microl of

neutralization buffer and mix

immediately and thoroughly by inverting

the tube 4 to 6 six times do not Vortex

the solution should become cloudy if

using a Cogen kit mix until all traces

of blue are gone and the solution is

colorless this neutralization buffer

contains potassium acetate and acetic

acid the potassium acetate Returns the

pH to neutral allowing the DNA strands

to renature the long single strands of

chromosomal DNA get tangled with other

cellular debris such as protein and

lipids to form an insoluble precipitate

meanwhile the smaller and still

intertwined plasma DNA strands quickly

rehybridize and remain in

solution now close the tubes and

centrifuge them for 10 minutes at full

speed remember to balance your

centrifuge while your samples are

centrifuging label one spin column for

each culture you started with each spin

column consists of the column set inside

an empty collecting tube label both both

the collecting tube and the spin column

after 10 minutes a white pellet should

appear at the bottom and side of the

microfuge tube this pellet consists of

the proteins lipids and genomic DNA that

precipitated out when you added the

neutralization buffer the plasma DNA

that you want is still in solution in

the

supernatant the supernatant is what you

want to keep add the clear supernatent

to the spin column by pipetting or

pouring the supernat directly into the

column you can now discard the microfuge

tube containing the pellet there is no

cap for the spin column so place it

carefully in a micro centrifuge remember

to balance the centrifuge spin for 1

minute at full speed during the spin

label clean 1.5 mil microfuge tubes to

hold your final purified plasma DNA be

sure to include your initials and the

date once the spin is done remove the

spin column colum and collecting tube

from the centrifuge dump out the flow

through that is now in the collecting

tube and place the column back in the

collecting

tube the spin column contains a silica

membrane that will tightly bind the

plasma DNA that is in the salty buffered

supernant any remaining RNA proteins or

other macromolecules pass through the

membrane and are found in the flow

through after

centrifugation at this point the DNA is

bound to the silica Matrix in the column

to remove the salts left over from the

buffer wash the spin column by adding

750 microl of wash buffer which contains

approximately 70% ethanol the salts are

soluble in 70% ethanol but the DNA is

insoluble centrifuge for 1 minute the

insoluble plasma DNA remains bound to

the silica membrane while the flow

through carries away the salt discard

the flow through residual ethanol from

the wash buffer May inhibit subsequent

enzymatic reactions so it is important

to dry the spin column with a Second

Spin centrifuge again for 1 minute to

remove any residual wash buffer when you

are done place the spin column in the

clean appropriately labeled 1.5ml

microfuge tube your plasma DNA is still

on the spin column discard The

Collection tube with any flow through

add 50 microl of uion buffer directly to

the membrane in the spin column let the

spin column stand 1 minute while the DNA

becomes

solubilized then centrifuge the colum

and tube for 1 minute the microfuge tube

caps will not close so be sure that the

Caps are facing toward the center of the

centrifuge so that they do not break off

while spinning after centrifugation you

should see about 50 microl of clear

colorless liquid in the bottom of your

microcentrifuge tube this contains

purified plasma DNA discard the spin

column but keep and store your tube of

purified plasma

DNA