MISS :Salsabeel AL Jou jou
1. To understand the basic procedures involved in the
isolation process of plasmid from bacteria.
2. To be understood the differences between two methods of
plasmid isolation.
3. To estimation Plasmid DNA quality and quantity.
4. To visualization separating DNA with gel electrophoresis
by staining with ethidium bromide.
5. To use plasmid in transformation after isolated from
bacterial cells.
Plasmids: vehicles of recombinant DNA
Bacterial cell
Non-chromosomal DNA
Replication: independent of the chromosome
Many copies per cell
Easy to isolate
Easy to manipulate
Plasmid DNA isolation
 Plasmid DNA isolation requires separation of this DNA
from the chromosomal DNA in the bacterial cell as well as
from the polysaccharides, lipids and proteins that constitute
the cell.
The various plasmid isolation techniques that are currently in
use can be divided into three phases:
 growth of microorganisms that contain the plasmid
 Harvesting , resuspension and lysis of bacterial cells.
 Separate plasmid DNA from contaminants .
 Purification of plasmid DNA by separating it from proteins
and chromosomal DNA.
Methods for Plasmid Isolation
Depending on amount of
plasmid to be isolate
Miniprep
Maxiprep
Depending on the principle
there are several method to
isolation plasmid
SDS-alkaline
denaturation
Rapid boiling
method
In our lab :• we used miniprep method (promega kit).
•
purification plasmid by solid phas purificatin
(with used silica based column )
•
learns Principle of SDS-alkaline denaturation.
Principle of the SDS-alkaline
denaturation method
Overnight Culture Suspension
• Pick a single colony and inoculate in 5
ml of LB containing 20 mg/l ampicilin
• Incubate overnight at 37oC
• The next day:
• Centrifuge 1.5 ml of broth containing
cells in a tube
• Discard supernatant
Resuspension solution (soln1)
 Resuspension of the bacterial pellet in buffer
containing glucose ,Tris ,EDTA
 Glucose :
Give osmotic shock that lead to rupture of cell wall and
membrane
 The ethylene diamine tetraacetate (EDTA) chelates
divalent metals (primarily magnesium and calcium)
then inhibits Dnases activity of cellular enzymes.
Tris is buffer substance
In addition to the above listed components, the
resuspension solution can also contain RNase enzyme in
order to break down ribonucleic acids.
alkaline lysis solution (soln2)
 Mix this by gentle inversion and incubate on
ice .
 This solution contain:
 1-SDS
 2-NaOH
 a. SDS (sodium dodecyl sulfate)
 Dissolves membranes (disintegrates the lipid
structure of the cell membrane)
 Binds to and denatures proteins
b. NaOH
 denatures both plasmid and chromosomal DNA to
single strand .
 Chromosomal DNA separate completely into single
strand but plasmid not fully separate and remain
entangled after denaturation.
solution 3 effects:
Potassium acetate / acetic acid solution
A-Acetic acid :Neutralizes NaOH
 plasmid DNA is allowed to renature. Sheared
cellular DNA remains denatured as single
stranded DNA (ssDNA).
 The ssDNA is precipitated, since large
ssDNA molecules
 Since plasmid DNA is small circular and
supercoiled, whereas genomic DNA is too
long to reaneal properly and remain single
strand .
 Potassium acetate:
 Converts soluble SDS to insoluble PDS
 the potassium salt of SDS is insoluble, so the
protein and detergent precipitate and
aggregate, which assists in the entrapment of
the
high-molecular-weight
chromosomal
DNA.
Separate plasmid DNA from contaminants
Separate plasmid DNA from contaminants by
centrifugation
•
Supernatant contains:
- Plasmid DNA
- Some cellular constituents
•
Sediment contains:
- PDS
- Lipids
- Proteins
- Chromosomal DNA
NOTE:
The soluble plasmid DNA (supernatant) is ready to be
further purified.
alkaline lysis
Alkaline
conditions
denature DNA
purify plasmid DNA using
silica-based columns
(Promega kit)
The PureYield™ Plasmid Miniprep System
 isolates high-quality plasmid DNA for use in eukaryotic
transfection and in vitro expression experiments.
 The system provides a rapid method to purify plasmid
DNA using a silica-membrane column.
 Plasmid DNA can be purified in less than 10 minutes,
depending on the number of samples processed, greatly
reducing the time spent compared to silica resin or other
membrane column methods.
 The PureYield™ Plasmid Miniprep System is designed to
purify 1.5–7.5μg of plasmid DNA .
This system can be used to isolate any plasmid from E. coli
hosts but works most efficiently when the plasmid is up to
20,000bp in size.
Note:
For best results, use plasmids that are 10,000bp or less.
The protocol presented in this Technical Bulletin describes
isolation of plasmid DNA from E. coli.
Plasmid yield will vary, depending on :
1. a number of factors,
2. culture volume,
3. plasmid copy number,
4. type of culture medium
5. bacterial strain used.
Component of kit: (30–37ºC )
• Cell Lysis Buffer (CLC) (Blue.
• Neutralization Solution (NSC).
• Endotoxin Removal Wash (ERB).
• Column Wash Solution (CWC).
• Elution Buffer (EBB).
Tris-HCl (pH 8.5) , EDTA.
• PureYield™ Minicolumns.
• PureYield™ Collection Tubes.
(store at 4-8 c)
Before you begin:
dilute the Column Wash Solution (provided) with 95%
ethanol.
PureYield™ Plasmid Miniprep System
Protocol :
1. Centrifugation Protocol.
2. vacuum Protocol.
3. Alternative Protocol for Larger Culture Volumes.
Protocol :- (Centrifugation Protocol.)
 Check the Cell Lysis Buffer to be sure that
components have not precipitated during shipping.
If precipitation has occurred, resuspend the buffer by
incubating the bottle at 30–37ºC for 30 minutes and
mixing by inversion.
*Note:• We recommend the use of LB medium.
• If you are using other growth media, pellet the cells
and resuspend the cell pellet in TE bufer or water prior
to lysis.
Perform the following procedure at room temperature.
1. Transfer 600μl of bacterial culture grown in LB
medium to a 1.5ml microcentrifuge tube.
2. Add 100μl of Cell Lysis Buffer, and mix by
inverting the tube 6 times.
*( The solution should change from opaque to clear blue, indicating
complete lysis)
3 . Add 350μl of cold (4–8°C) Neutralization Solution,
and mix thoroughly by inverting the tube.
Note:The sample will turn yellow when neutralization is
complete, and a yellow precipitatewill form.
Invert the sample an additional 3 times to ensure
complete neutralization.
4. Centrifuge at maximum speed in a microcentrifuge for
3 minutes.
5. Transfer the supernatant (~900μl) to a PureYield™
Minicolumn.
Note:Do not disturb the cell debris pellet. For maximum
yield, transfer the supernatant with a pipette.
6. Place the minicolumn into a PureYield™ Collection
Tube, and centrifuge at maximum speed in a
microcentrifuge for 15 seconds.
7. Discard the flowthrough, and place the minicolumn
into the same PureYield™ Collection Tube.
8. Add 200μl of Endotoxin Removal Wash to the
minicolumn. Centrifuge at maximum speed in a
microcentrifuge
9.Add 400µl of Column Wash Solution to the minicolumn.
Centrifuge at maximum speed in a microcentrifuge for 30
seconds.
10. Transfer the minicolumn to a clean 1.5ml microcentrifuge
tube, then add 30µl of Elution Buffer directly to the
minicolumn matrix. Let stand for 1 minute at room
temperature.
Notes:
Nuclease-free water at neutral pH can also be used to elute
DNA.
11. Centrifuge at maximum speed in a microcentrifuge for 15
seconds to elute the plasmid DNA. Cap the microcentrifuge
tube, and store eluted plasmid DNA at –20°C.
If the cells was cultured in L.B media
you can tack 600M and put cell lyses
directly , without harvesting by
centrifuge
Harvest cells by centrifugation
, if cells was cultured in other
growth media
*Spin ~5,000 rcf
Supernatant
(clear)
Pelleted cells
resuspend the cell
pellet in TE bufer.
or water prior to lysis
Discard
supernatant
Supernatant
(Plasmid DNA ,Soluble
cellular constituents)
pellet
Protein , lipid,
chromosomal DNA)
Inverted until the
color turn to yallow
Add 600M cell culture
add 100M lysis solution
Centrifuge 3min
Inverted and add 350M
neutralization solution
Centrifuge 15s
Transfer supernatant
to Minicolumn
Centrifuge 15s
Discard the flowthrough, and 200μl of Endotoxin
place the minicolumn into the
Removal Wash
same Collection Tube
Note:- we will use on column only
400µl of Column
Wash Solution
Centrifuge
30s
Transfer the column
in apendrof tube
Centrifuge
30s
Add 30µl of Elution
Buffer
Pure plasmid
General Instructions in procedure
 DNA is very sensitive to mechanical stress, therefore
shearing forces caused by mixing/vortexing or fast
pipetting must be avoided as soon as cell lysis occurs.
 All mixing steps during and after cell lysis should be
performed carefully by inverting the tubes several times
(8-10 fold).
 Gloves should be worn in order to prevent contamination
with Dnases Autoclaved (DNase-free) buffer solutions,
tubes and tips should be used.
 For long-term storage, plasmid DNA should be frozen in
aliquots of storage TE buffer. Repeated thawing and
freezing of DNA should be avoided.
Finally(practical quize):1. Each student has to extracted plasmid alone
by follow the promega Kit.
2. Check the quality of your extracted plasmid in
nanodrope instrument.
3. Preaper1% Agarose gel , and loading your
sample.
Good Luke