Isolating Plasmid DNA
Summary
TLDRThis 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
🧬 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.
🔬 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.
🧪 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.
Mindmap
Keywords
💡Plasmid
💡Selectable marker
💡Origin of replication
💡Multiple cloning site (polylinker)
💡Transformation
💡Antibiotic selection
💡Plasmid isolation kit
💡Lysis buffer (L buffer)
💡Neutralization buffer
💡Spin column
💡Elution buffer
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
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