Preparation of plasmid DNA from bacteria
Content
Introduction ....................................................................................................................................... 2
Materials............................................................................................................................................ 4
Collection of bacteria ......................................................................................................................... 5
Destruction of bacteria....................................................................................................................... 6
Separation of plasmid DNA from genomic DNA and cell debris ......................................................... 7
Purification of plasmid DNA ............................................................................................................... 8
Elution of purified plasmid DNA ......................................................................................................... 9
Estimation of the concentration of the isolated plasmid-DNA ........................................................... 10
Verification via agarose gel electrophoresis..................................................................................... 11
Protocol: preparation of plasmid DNA .............................................................................................. 13
1
Preparation of plasmid DNA from bacteria
Introduction
Nucleic acids can be isolated and purificated from all different types of organisms and cell
material(s). Some options are shown below (fig.1). It is possible to isolate DNA and RNA.
Plasmid DNA may be isolated from bacteria or yeast. Depending on the amount of cell material, the
isolation is called mini–, midi–, or maxi-preparation.
Genomic DNA or RNA may be isolated either from plants, funguses or animals. Very common is the
isolation of DNA from blood cells such as lymphocytes or the isolation of DNA from various types of
tissue and cells.
It is also possible to purify DNA after a polymerase chain reaction or after an agarose gel
electrophoresis.
2
Preparation of plasmid DNA from bacteria
The following flow chart (fig. 2) shows the isolation of plasmid DNA from small scale bacterial
cultures.
Step 1
Step 2
Step 3
Step 4
Step 5
Fig. 2: Isolation of plasmid DNA from bacteria,
mini preparation
Bacteria of an over night culture are pelleted by centrifugation in order to separate them from liquid
media. In step 1 the pelleted bacteria are treated with a lysis buffer containing alkali and sodium
dodecyl sulfate. The cell walls and membranes are destroyed. Genomic DNA denaturates, while the
plasmid DNA remains, due to its super coiled conformation for the most part in its natural
conformation.
In step 2 the bacteria are centrifugated. Genomic DNA and cell debris are pelleted. The plasmid
DNA stays in solution.
In step 3 the supernatant which contains the plasmid DNA is retained and passed through a spin
filter tube. The nucleic acids bind specifically to the surface of the glass fibers of the filter.
In step 4 the bound DNA is purified from salt, proteins and other cellular impurities by passing
washing buffers trough the filter.
Finally in step 5 the purified plasmid DNA can be obtained by elution with low-salt buffer.
3
Preparation of plasmid DNA from bacteria
Materials
For the preparation you need …
9
3
6
4
1
2
7
8
5
Fig. 3: Materials for a mini preparation
1 approximately 1 – 5 ml of a bacteria culture if isolating high copy plasmids,
2 a table top centrifuge,
3 a clock,
4 an autoclavable flask for fluid bacteria waste,
5 a set of the different detergents and buffers,
6 the suspension buffer and the binding buffer have to be placed on ice,
7 sterile 1.5 ml reaction tubes,
8 a filter tube which binds the nucleic acids and a collection tube.
Furthermore you need a centrifuge in order to pellet the bacteria culture, a set of micropipettes and
sterile tips for amounts in between 200 and 1000 µl. Sterile tips should be used to avoid
contamination of the isolated DNA.
9 You should wear latex gloves for protection: Some of the detergents contain alkali.
A variety of commercial kits is available, which provide all solutions and filter tubes. Their
compositions may slightly differ but have more or less the same tasks.
The suspension buffer is slightly alkaline and contains RNAse in order to destroy liberated RNA.
The alkaline lysis buffer contains Sodium Hydroxide. Sodium Dodecyl Sulfate destroys the cell
walls of bacteria, Sodium Hydroxide denatures proteins and genomic DNA, small supercoiled
Plasmid DNA is less affected. The binding buffer is slightly acidic. The high amount of potassium
acetate leads to the precipitation of the denaturated proteins. Denaturated DNA renaturates. Due to
its size genomic DNA tends to renaturate not accurately and will precipitate together with the
proteins. The small sized denaturized plasmid DNA renaturates and stays in solution. The process
of precipitation may be supported by using chilled buffer. As mentioned, keep it on ice. Two
washing buffers eliminate all debris, which might have bound to the filter besides the plasmid DNA.
The low salt elution buffer elutes the purified DNA from the filter.
4
Preparation of plasmid DNA from bacteria
Collection of bacteria
For collection of bacteria …
1. pellet the bacterial cells of an overnight bacteria culture by centrifugation of 5 minutes at
approximately 4000 revolutions per minute.
2. In order to stabilize the centrifuge, do not forget to add a tube of the same weight opposite of the
tube containing the bacteria,
3. Depending on the type of centrifuge and the height /size of the tubes, the caps have to be taken
off before centrifugation.
4. After centrifugation you should be able to distinguish the cleared liquid media and the bacteria
pellet.
2
1→
Fig 4: Pelleted bacteria (1) and cleared liquid media (2)
5. Discard the supernatant into the waste container
6. Dab the last amount of liquid on a paper towel. Any amount of liquid media may reduce your
result. The bacteria pellet will remain in the tube.
Fig 5:
Removing last amounts of liquid
5
Preparation of plasmid DNA from bacteria
Destruction of bacteria
8. Add 250 µl suspension buffer to the pellet.
The suspension buffer is slightly alkaline and contains RNAse in order to destroy liberated RNA
later in the process.
9. Resuspend the bacterial pellet and mix well by pipetting up and down or vortexing.
Fig 6: Suspension buffer is added to the bacterial pellet
10. You may transfer the suspension into a small reaction tube.
11. Add 250 µl of lysis luffer to the resuspended pellet.
As mentioned the lysis buffer is alkaline. It contains Sodium Dodecyl Sulfate and Sodium
Hydroxide. Sodium Dodecyl Sulfate destroys the cell walls of bacteria, Sodium Hydroxide
denatures proteins and genomic DNA. Small supercoiled plasmid-DNA is less affected.
12. Mix gently by carefully inverting the tube 3 to 6 times. Do not vortex to avoid shearing of
genomic DNA.
13. Incubate for 5 minutes at room temperature.
When opening the reaction tube carefully after 5 minutes, you may discover a fine file of released
genomic DNA. The file usually appears between the neck of the tube and its` lid.
Fig 7: File of genomic DNA
6
Preparation of plasmid DNA from bacteria
Separation of plasmid DNA from genomic DNA and cell debris
14. Add 350 µl of chilled binding buffer
15. Mix gently by inverting the tube 3 to 6 times.
The binding buffer is slightly acid. The high amount of potassium acetate leads to the
precipitation of the denaturated proteins. Denatured DNA renaturates. Due to its size genomic
DNA tends to renature not accurately and will also precipitate. The small sized denatured
plasmid DNA renaturates and stays in solution.
Fig 8: Mixing by inverting the reaction tube
16. Incubating the tube on ice for 5 minutes will accelerate the process.
17. Centrifuge for 10 minutes at 13000 rpm in the table top centrifuge.
18. After centrifugation you will discover a white or greyish pellet – cell debris and genomic DNA.
←
Fig. 9: Precipitated cell debris
and genomic DNA before
centrifugation
Fig. 10: Pelleted cell debris and genomic DNA (←) after
centrifugation
7
Preparation of plasmid DNA from bacteria
19. Transfer the entire supernatant carefully into the filter tube. It contains plasmid-DNA. The filter
tube should be placed into a collection tube. Avoid transferring any pellet.
20. Insert the entire tube assembly into the centrifuge and spin 1 minute at 13000 revolutions per
minute. While the buffer passes the filter, the nucleic acids bind specifically to the surface of the
glass fibers.
Purification of plasmid DNA
After centrifugation the collection tube contains the binding buffer, the plasmid DNA is bound to the
filter surface.
21. Remove the filter tube from the collection tube, discard the flow through liquid.
Reinsert the filter tube in the same collection tube.
22. Add 500 µl of the first washing buffer to the filter tube. The washing buffer eliminates all salts,
proteins and other cellular impurities, which might have bound to the filter besides the plasmid
DNA.
23. Centrifuge again for 1 minute at top speed.
24. Discard the flowthrough.
1→
←2
Fig 11: Washing step I,
the flow through is discarded,
filter tube (1), collection tube (2)
25.
26.
27.
28.
For a second washing step add 700 µl of the second washing buffer to the filter tube.
Centrifuge for 1 minute at top speed.
Again discard the flowthrough.
Centrifuge the entire tube assembly for an additional minute at top speed.
This will eliminate any leftovers of washing buffer.
8
Preparation of plasmid DNA from bacteria
Elution of purified plasmid DNA
29. Discard the collection tube and insert the filter tube into a clean sterile 1,5 reaction tube.
30. Add 100 µl of elution buffer into the filter tube.
The low salt elution buffer elutes the purified DNA from the filter
31. Centrifuge the entire assembly for 1 minute at full speed.
3→
2→
←1
Fig 12: The filter tube (1) containing the plasmid DNA is inserted
into a clean sterile reaction tube (2), the collection
tube (3) will be discarded
32. The filter tube is discarded.
The reaction tube now contains the purified plasmid DNA.
←1
↑2
Fig 13: The filter tube (1) is discarded; the reaction tube (2)
contains the purified plasmid DNA
9
Preparation of plasmid DNA from bacteria
Estimation of the concentration of the isolated plasmid-DNA
The concentration of the purified DNA can be estimated by measuring the absorbance at 260 nm in
an UV-photometer. An absorption of 1 stands for a concentration of approximately 50 μg double
stranded DNA per ml. From there the concentration of a DNA solution can easily be calculated.
A sufficient DNA purity is reached if the ratio of the absorbance measured at 260 nm and measured
at 280 nm is 1.8 to 2.0.
1. At first the blank value is prepared. Use water or the same dissolvent you have used for your
DNA. Fill it into a cuvette. In this case deionized water is used.
2. Prepare a dilution of your isolated DNA. Usually dilutions in between 1:10 or 1:100 are
appropriate.
The dilution can be directly done in the cuvette. First pipette dissolvent into the cuvette, then add
the DNA.
3. Mix the solvent by pipetting up and down.
4. For the zero measurement the blank value is inserted into the photometer, which should be just
the solvent without DNA.
After setting the photometer the display should show an absorption of “Zero”.
5. Now the DNA-sample can be measured.
Fig 14: Inserting a cuvette into the UV-photometer
10
Preparation of plasmid DNA from bacteria
Verification via agarose gel electrophoresis
You can check if the right DNA has been isolated and purified by performing a restriction and
agarose gel electrophoresis
1. For a 20 ul restriction add sufficient volumes of the following solutions into a clean an sterile
reaction tube:
- deionized water
- restriction buffer, which is corresponding to the used restriction enzyme
- DNA- solution, holding approximately 1 μg of DNA
- and a suitable restriction enzyme. Restriction enzymes are temperature-sensitive,
they should be always kept on ice.
2. Mix gently.
3. A short centrifugation afterwards guarantees that all compounds are united at the bottom of the
reaction tube.
4. Incubate the tube for one hour at 37 °C.
5. After the restriction reaction spin the tube briefly in the centrifuge. Condensate might have
developed in the inside of the lid of the tube.
6. Add a sufficient amount of DNA probe buffer. It will stop the reaction.
7. A brief centrifugation unites all compounds at the bottom of the reaction tube.
Additionally you might also centrifuge the DNA-ladder.
8. Then the samples – the restricted DNA and the DNA ladder - are carefully loaded into the slots
of the submerged agarose gel.
9. Close the lid of the gel tank and apply a voltage of app. 100 V. Make sure that the DNA migrates
towards the anode.
10. Depending on the size of the gel, it will run from approximately 30 minutes to one hour.
Fig 15: Loading the samples into the slots of the agarose gel
11
Preparation of plasmid DNA from bacteria
The photography shows the result of a gel electrophoresis.
The DNA has been stained with ethidiumbromide*.
cathode
(-)
← slots
Lane 1 shows the restricted plasmid DNA. It has been
cleaved twice by the restriction enzyme.
Lane 2 shows the DNA ladder with the following fragments:
10000 bp
8000 bp
6000 bp
5000 bp
4000 bp
3000 bp
2500 bp
2000 bp
1500 bp
1000 bp
500 bp
anode
(+)
1
2
Fig 16: Agarose gel electrophoresis
*Notice: When working with ethidiumbromide nitrile gloves should be worn.
12
Preparation of plasmid DNA from bacteria
Protocol: preparation of plasmid DNA
Materials
For a mini preparation of plasmid DNA you need
 approximately 1 – 5 ml of a bacteria culture, if isolating high copy plasmids
 a centrifuge in order to pellet the bacteria culture,
 a table top centrifuge
 a clock
 an autoclavable flask for fluid bacteria waste,
 a set of the different detergents and buffers*
 sterile 1,5 ml reaction tubes
 a filter tube which binds the nucleic acids and a collection tube
 a set of micropipettes and sterile tips for amounts in between 200 and 1000 µl
 latex gloves for protection
* Commercial kits usually contain all needed detergents and buffers. Most kits contain
 suspension buffer
 lyses buffer
 binding buffer
 washing buffer I
 washing buffer II
 elution buffer
Collection of bacteria
1.
2.
3.
4.
5.
Pellet the bacterial cells of an overnight bacteria culture by centrifugation of 5 minutes at
approximately 4000 revolutions per minute.
In order to stabilize the centrifuge, do not forget to add a tube of the same weight opposite of
the tube containing the bacteria,
Depending on the type of centrifuge and the height /size of the tubes, the caps have to be taken
off before centrifugation.
After centrifugation you should be able to distinguish the cleared liquid media and the bacteria
pellet.
Discard the supernatant into the waste container. Dab the last amount of liquid on a paper
towel.
Destruction of bacteria
6.
7.
8.
9.
10.
Add 250 µl suspension buffer to the pellet.
Resuspend the bacterial pellet and mix well by pipetting up and down or vortexing.
You may transfer the suspension into a small reaction tube.
Add 250 µl of lysis buffer to the resuspended pellet.
Mix gently by carefully inverting the tube 3 to 6 times. Do not vortex to avoid shearing of
genomic DNA.
11. Incubate for 5 minutes at room temperature.
13
Preparation of plasmid DNA from bacteria
Separation of plasmid DNA from genomic DNA and cell debris
12. Add 350 µl of chilled binding buffer
13. Mix gently by inverting the tube 3 to 6 times.
14. Incubate the tube on ice for 5 minutes.This will accelerate the process.
15. Centrifuge for 10 minutes at 13000 rpm in the table top centrifuge.
16. After centrifugation you will discover a white or greyish pellet – cell debris and genomic DNA.
17. Transfer the entire supernatant carefully into the filter tube, which should be placed into a
collection tube. Avoid transfering any pellet.
18. Insert the entire tube assembly into the centrifuge and spin 1 minute at 13000 revolutions per
minute. The Plasmid-DNA will attach to the filter.
Purification of Plasmid DNA
19. Remove the filter tube from the collection tube, discard the flow through liquid.
Reinsert the filter tube in the same collection tube.
20. Add 500 µl washing buffer I to the filter tube. Centrifuge again for 1 minute at top speed.
21. Discard the flowthrough.
22. For a second washing step add 700 µl Washing Buffer II to the filter tube.
23. Centrifuge for 1 minute at top speed.
24. Again discard the flowthrough.
25. Centrifuge the entire tube assembly for an additional minute at top speed.
This will eliminate any leftovers of washing buffer.
Elution of purified plasmid DNA
26. Discard the collection tube and insert the filter tube into a clean sterile 1, 5 reaction tube.
27. Add 100 µl of elution buffer into the filter tube.
Centrifuge the entire assembly for 1 minute at full speed.
28. The filter tube is discarded.
The reaction tube now contains the purified plasmid DNA.
14