Laboratory Protocols
Plasmid DNA isolation
For this practical, a strain of E. coli with a GFP-gene on its plasmid is required. This can
be, for instance, the pGLO-plasmid, available in the Bio-Rad kit. This is a starter kit, which
also contains all kinds of disposables and media.
The plasmid pGFP-uv or another plasmid is also appropriate and may be available
somewhere in your own institute or a nearby institute.
Growth of a culture of the E. coli with the appropriate plasmid is prerequisite, and a
protocol is given below. This is followed by protocols for isolation of the plasmid DNA from
the E. coli in two different ways.
Prerequisite: Growing Bacteria in Liquid Media
Materials
1. LB medium: 10 g bacto tryptone and 5 g bacto yeast extract in 10 g NaCl are dissolved
in 900 ml of demineralised water. After correcting the pH to 7.0, and adjusting the
volume to 1000 ml, the medium is sterilised for 20 minutes at 121 °C. After sterilisation
and cooling down the medium (to below 50 °C), ampicillin (stock solution: 50 mg / ml) is
added to the medium with a final concentration of 80 µg ampicillin per ml medium.
2. E. coli strain with the plasmid pGFPuv or pGLO for the production of GFP protein.
Procedure
1. Inoculate 20 ml LB medium with the E. coli pGFPuv or pGLO strain approximately 16
hours before the start of the isolation of plasmid DNA.
2. The culture is cultivated at 37 °C under aeration with air (by rotating the vessels with
medium at 200 rpm).
3. Before starting with the plasmid DNA isolation, the culture (with bacteria; how can you
see if there has been bacterial growth?) is cooled in ice/water bath for 15 minutes.
4. Take from the culture 200 µl and dilute this with 1800 µl fresh LB medium. Measure the
optical density at 660 nm (use LB medium as a blank).
Extinction = ……; dilution factor = …
Calculate the amount of cells in cells/ml (Extinction 660 nm = 1 corresponds to 10 9
cells/ml).
Laboratory Protocols
A. QIAGEN PROTOCOL
DNA Isolation
Purpose
To isolate plasmid DNA on a small-scale - 1 to 20 µg plasmid DNA - two methods will be
referred to: that with the use of the Qiagen prep kit, and the alkaline lysis method.
The plasmid might be isolated from E. coli cells using a Qiagen Qiaprep Miniprep kit. The
following procedure has been adapted from the Qiagen Protocol.
The QIAprep Principle
The QIAprep miniprep procedure is based on alkaline lysis of bacterial cells followed by
adsorption of DNA onto silica in the presence of high salt. The unique silica-gel membrane
used in QIAprep Miniprep Kits completely replaces glass or silica slurries for plasmid
minipreps.
The procedure consists of three basic steps:
• Preparation and clearing of a bacterial lysate
• Adsorption of DNA onto the QIAprep membrane
• Washing and elution of plasmid DNA
All steps are performed without the use of phenol, chloroform, CsCI, ethidium bromide,
and without alcohol precipitation.
Alkaline lysis of bacteria
The QIA-prep miniprep procedure uses the modified alkaline lysis method of Birnboim &
Doly. Bacteria are lysated under alkaline conditions, and the lysate is subsequently
neutralised and adjusted to high-salt binding conditions in one step, ready for purification
on the QIA-prep silica-gel membrane. For more details on growth of bacterial cultures and
alkaline lysis, please refer to original QIA-prep handbook.
Lysate clearing
In the ‘QIA-prep Spin’ the lysates are cleared by centrifugation.
DNA adsorption to the QIAprep membrane
QIA-prep columns, strips, and plates use a silica-gel membrane for selective adsorption of
plasmid DNA in high-salt buffer and elution in low-salt buffer. The optimised buffers in the
lysis procedure combined with the unique silica-gel membrane ensure that only DNA will
be adsorbed, while RNA, cellular proteins, and metabolites are not retained on the
membrane but are found in the flow-through; see figure below.
Laboratory Protocols
Materials
1.
2.
3.
4.
5.
6.
7.
8.
9.
Disposals: sterile vials, sterile pipette tips
Micro pipettes
Micro centrifuges
Buffer P1: 50 mM Tris-HCl, pH 8.0, 10 mM EDTA and 100 µg RNase A per ml
Buffer P2: 200 mM NaOH and 1 % SDS
Buffer N3: guanidine HCl and HAc, pH 4.5
Buffer PB: with guanidine HCl and isopropanol
Buffer PE: with ethanol
Buffer EB: 10 mM Tris-HCl, pH 8.0, 1 mM EDTA
Laboratory Protocols
Method
Notes / questions
1. The yield of plasmid DNA is much higher if the bacteria are totally re-suspended.
2. Antibiotics and medium additives, such as vitamins, are highly thermo-instable; these
solutions must be filtered sterilised (0.2 µm) and are stored in a freezer (- 40 °C) as a
stock solution (10 up to 1000 times concentrated)
3. The quality of water for solutions is utmost important; sterile MQ water is normally
used.
4. What are the functions of Tris-HCl, EDTA, RNase A, NaOH, SDS, guanidine HCl, HAc,
isopropanol and ethanol?
5. What is the function of the silica column?
6. Why is the DNA solution stored preferentially in buffer EB (see above) and not in sterile
MQ water?
7. Control actions:
- Buffer P1 must contain RNase,
- Check Buffers P2 and N3 before use for salt precipitation. Redissolve any precipitate
by warming to 37 °C. Do not shake vigorously!
- Buffer PE must contain ethanol
8. All centrifugation steps are carried out at maximum speed (13,000 rpm) in a
conventional microcentrifuge.
Buffers P2, N3 and PB contain irritants. Wear gloves when handling these buffers.
Using a microcentrifuge
This protocol is designed for purification of up to 20 µg of high-copy plasmid DNA from 1
up to 5 ml overnight cultures of E. coli in LB (Luria-Bertani) medium. For purification of
low-copy plasmids and cosmids, large plasmids (>10 kb), and DNA prepared using other
methods, some modifications must be applied.
Procedure
1. Resuspend the pellet of bacterial cells in 250 µl of Buffer P1 and transfer to a
microfuge tube.
Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible
after resuspending the pellet.
2. Add 250 µl of Buffer P2 and gently invert the tube 4-6 times to mix.
Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic
DNA. lf necessary, continue inverting the tube until the solution becomes viscous and
slightly clear. Do not allow the lysis reaction to proceed for more than 5 minutes.
3. Add 350 µl of Buffer N3 and invert the tube immediately but gently 4-6 times. To avoid
localised precipitation, mix the solution gently but thoroughly, immediately after addition
of Buffer N3. The solution should become cloudy.
4. Centrifuge for 10 min.
A compact white pellet will form.
During centrifugation, place a QIA-prep spin column in a 2-ml collection tube.
5. Apply the supernatants from step 4 to the QIA-prep column by decanting or pipetting.
6. Centrifuge 30-60 sec. Discard the flow-through.
7. (Optional): Wash QIA-prep spin column by adding 0.5 ml of Buffer PB and centrifuging
30-60 sec. Discard the flow-through.
This step is necessary to remove trace nuclease activity when using end-A + strains
such as the E. coli JM series, HB 101 and its derivatives, or any wild-type E. coli strain,
Laboratory Protocols
which have high levels of nuclease activity or high carbohydrate content. Host strains
such as XL-1 Blue and DH5 do not require this additional wash step.
8. Wash QIA-prep spin column by adding 0.75 ml of Buffer PE and centrifuging 30-60
sec.
9. Discard the flow-through, and centrifuge for an additional 1 min to remove residual
wash buffer.
IMPORTANT: Residual wash buffer will not be completely removed unless the
flow-through is discarded before this additional centrifugation. Residual ethanol from
Buffer PE may inhibit elution from the column and subsequent enzymatic reactions.
10. Place QIA-prep column in a clean, sterile 1.5-ml microfuge tube. To elute DNA, add 50
µl of Buffer EB (10 mM Tris-Cl, pH 8.5) or H2O to the centre of each QIA-prep column,
let stand for 1 min, and centrifuge for 1 min.
Repeat this step by adding again 50 µl of Buffer EB (10 mM Tris-Cl, pH 8.5) or H2O to
the centre of each QIA-prep column, let stand for 1 min, and centrifuge for 1 min.
11. Take from the obtained DNA solution (approximately 100 µl) 20 µl and add 1980 µl
sterile MQ water; so the dilution factor = ….
12. Transfer this diluted DNA solution to a quartz optical cell and measure the optical
density at 260 and 280 nm in a spectrum range from 200 up to 400 nm (see for more
details: protocol 3: measurement of DNA and RNA)
13. The undiluted DNA solution should be coded and stored in a freezer.
Questions / tasks:
1. Calculate the DNA concentration of the diluted and undiluted DNA solutions.
A DNA solution of 1 mg DNA / ml gives an optical density of 20.0 at 260 nm.
2. Calculate the yield of total DNA in µg; so the undiluted solutions contains …. µg / µl.
3. How many ml’s of cultivated bacteria are used for the DNA isolation; this means that,
per ml cultivated bacteria, …. µg DNA is isolated.
4. After correction for the amount of bacteria cells per ml and the optical density of the
broth you get a DNA yield of … µg per bacterium cell.
Answers to notes/questions:
3. The function of the following solutions/chemicals:
Tris-HCl: buffer solution
EDTA: binds 2+ ions like Magnesium
Rnase: breaks down the RNA in the cell
NaOH: (temporarily) denatures DNA
SDS: soap-like solution, dissolves membranes and denatures proteins
Guanidine HCl: removes traces of nuclease activity
HAc: precipitation of SDS-cell wall protein complexes, neutralisation of the (NaOH) solution
Isopropanol: aggregates plasmid DNA
Ethanol: wash step, removal of isopropanol and rests of salts
4. The function of the silica column is:
5. EB contains EDTA and Tris
Laboratory Protocols
B. Alkaline lysis method
Notes / questions
1. Make a work schedule. Illustrate where your plasmid will be at each point in the
procedure. The principle of the method is explained here; match this with the steps of
the protocol.
2. TEG (Tris, EDTA glucose solution) with RNase breaks down the bacteria in isotonic
environments. RNase specifically breaks down the RNA in the cell
3. SDS (sodium dodecyl sulphate, a soap-like solution) dissolves the membranes and
denatures the proteins, NaOH (temporarily) denatures the DNA.
4. Ice cold 7.5 M ammonium acetate precipitates the SDS-protein and cell wall remains.
This is also a neutralisation step, DNA can partially renature. Chromosomal DNA will
not be able to renature easily (because of its large size) and will be removed by
centrifugation. Plasmids are smaller, and will be renatured and stay in the supernatant.
5. The supernatant is transferred to a new Eppendorf tube, and isopropanol (isopropanol
can be replaced by twice the volume of 96% ethanol) is added in order to let the
plasmids aggregate. The washing with ethanol is meant to remove salts and remaining
isopropanol.
TE-buffer is the ideal solution to keep the DNA in, because the EDTA in it complexes
magnesium ions that the enzyme DNase needs to work.
Material
250 ml 10 % SDS solution
100 ml 10 M NaOH
250 ml 70% ethanol
100 ml 7.5 M ammonium acetate pH 7.8
100 ml isopropanol
100 ml TE buffer pH 8.0
100 ml STE-buffer pH 8.0
50 ml o.n. (= over night) culture of your E. coli with the pGLO or pGFP-uv plasmid
100 ml TEG pH 8.0
RNase
Ice
Eppendorf centrifuge and Eppendorf tubes of 2 ml and 1.5 ml
Pasteur pipettes
Pipettes and disposable tips 1-5 ml, 100-1000 µl.
Vacuum desiccator
Method
1. Put the 7.5 M ammonium acetate solution on ice
2. Make 1 ml of fresh NaOH 0.2 M, SDS 1% solution by mixing the following solutions (in
this order):
• 20 µl 10 M NaOH
• 880 µl milliQ water
• 100 µl SDS 10%
3. Spin 1.5 ml o.n. culture of the bacteria 1 minute by 12000 rpm (Eppendorf centrifuge).
4. Discard the supernatant and fill again with 1.5 ml o.n. culture. Centrifuge, discard the
supernatant and repeat this step again. Finally, you will have the pellet of 4.5 ml o.n.
culture on the bottom of your Eppendorf tube.
5. Wash the pellet by resuspension in 0.5 ml STE.
6. Spin for 1 minute at 12,000 rpm. Discard the supernatant.
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7. Resuspend the pellet in 200 µl TEG 100 µg/ml RNase. This step is crucial as total
resuspension must be achieved in order to succeed in the isolation of the plasmid!
8. Incubate 5 minutes at room temperature.
9. Add 400 µl of your freshly made NaOH 0.2M SDS 1 % solution and mix by hand.
10. Leave this for 5 to 10 minutes on crushed ice until you have a clear solution.
11. Add 300 µl of ice cold 7.5 M ammonium acetate at pH 7.8 and mix well.
12. Leave for 10 minutes in ice water.
13. Spin for 5 minutes at 13,000 rpm in the Eppendorf centrifuge.
14. Transfer the clear supernatant over to a new Eppendorf tube and add 0.6 x isopropanol
by volume. Mix by swirling and leave for 5 minutes at room temperature.
15. Centrifuge for 5 minutes at maximum rpm (Eppendorf centrifuge) and discard the
supernatant with a pasteur pipette with a capillary ending.
16. Wash the pellet with 1 ml 70% ethanol and spin 2 minutes at maximum rpm in the
Eppendorf centrifuge. Remove the ethanol; be careful not to remove the barely visible
pellet. This will not be totally at the bottom but somewhat on the side (due to the
centrifugal force) of the Eppendorf tube.
17. Remove the ethanol with a pasture pipette with a capillary ending. Let the rest of the
alcohol damp away: use a vacuum desiccator for instance. The pellet should not
become totally dry, because the last step is to redissolve it in 25 µl TE-buffer. While
adding the TE-buffer, be careful to let it flow over the pellet (with the DNA) in order to
get the viscous, DNA-containing solution. Spin for 3 minutes at maximal rpm, then save
on ice for instant use or put it in the freezer.