Practical Electives for Foreigners 2010

Practical Electives for Foreigners 2010
Practical Electives for Foreigners 2010
Date and Time: October 11, 2010, 12:15
Place: Laboratory A 51.1
Dr. Alexa Kabiersch
Fundamentals of microscopic anatomy
Topics
Basics of Microscopy
Which types of microscopes are used in Biology?
Practical Exercise:
How is a standard light transmission microscope adjusted?
Basics of Microscopic Anatomy
Which basic tissue types do we find in the human body?
Organs are made of several types of tissues. How are they analyzed?
Basics of Histological Techniques
How are different types of histological specimens prepared?
Demonstration of a tissue processor, a microtome and a staining bench
Practical Exercise:
Microscopic analysis of selected organs with focus on normal histology.
(The pathological changes of these organs will be studied in the first session of
Practical Pathophysiology)
Organs to be studies will be
lung
liver
blood smear
Reference (available in the library, absolutely necessary for preparation!)
Wheater's functional histology. A text and color atlas
Churchill Livingstone Elsevier, 4. or 5. ed.
Signatur: VVL1018
Morphology and structure of cells of the oral mucosa
As an example for an animal eukaryotic cell cells of the oral mucosa will be
examined. As animal cells generally are difficult to analyse in the microscope a
special staining technique has to be employed. The Giemsa stain specifically binds
the phosphate groups of DNA, preferably to such areas with a high amount of
Adenine-Thymine Bonding.
Material
Wooden spatula, microscope slides, methanol, Giemsa stain, A. dest., heating plate,
staining rack and staining cuvettes
Procedure
Scrap the inside of your cheek using a sterile spatula and transfer the smear onto a
microscope slide (label with your name – use a pencil!)
Let it air-dry at 37 °C (heating plate). The smear should look thick and grey.
Fix slides in methanol 5-7 minutes.
Let Air dry at 37 °C (heating plate).
Dilute Giemsa Stain 1:20 with A. dest.
Stain film for 15-60 minutes.
Rinse in deionized water.
Let Air dry and evaluate.
What is the size ratio of nucleus and cell – how is the nucleus positioned within the
cell?
Compare overall cell morphology to that of the Allium cepa epidermis cells.
Prepare a drawing.
Osmotic effects in erythrocytes
Cells are separated from their environment by a cytoplasm membrane (Please be
aware that bacterial, fungal or plant cells are additionally surrounded by cell walls).
The cytoplasm membrane basically consists of a lipid belayed with a hydrophilic
exterior and a hydrophobic interior part. However, this double belayed is not a
complete barrier: since the cell must be able to maintain an exchange with the
environment nutrients, oxygen etc. must be taken up by the cell, and waste-products
must be excreted. While hydrophobic substances and gases pass the membrane
easily, for larger substances or for those which are hydrophilic special transport
proteins are required.
Due to the selective permeability of the membrane cells may experience osmosis
when placed in a non-isotonic medium. Osmosis results from the net movement of
water from a region of low solute concentration to a region of high solute
concentration. If a cell is placed in a hypertonic medium, i.e. one in which the
concentration of solutes is higher than inside the cell, water will effuse the cell,
causing it to shrink. Placed in a hypotonic medium in which the extracellular
concentration of solutes is lower than inside the cell, water will enter the cell. As a
consequence the cell will swell or even burst.
Determination of osmoresistance of human erythrocytes is a diagnostic tool – defects
in the membrane structure will cause a decrease in resistance, while defects in
haemoglobin are known to increase resistance of the erythrocyte. Please mind
however, that the avian or sheep erythrocytes with which you are working here are
will differ from human erythrocytes – most notable for avian erythrocytes in the
presence of a nucleus, which is missing in human erythrocytes (and sheep
erythrocytes)
In this experiment the effect of hypotonic solutions on avian erythrocytes will be
studied via photometric analysis.
Therefore, erythrocytes will be placed in a series of solutions of various
concentrations of NaCl. The amount of hemoglobin released into the supernatant
after bursting of the cell is related to the concentration of solute in the environment.
Material
1.5 mL Eppendorff cup containing avian erythrocyte suspension in buffer, A. dest.,
NaCl stock solution (0.155 M), micropipette (100 µL), micropipette (1000 µL), blue
and yellow tips, 1.5 mL Eppendorff cups, cuvettes and cuvette rack, microcentrifuge,
photometer
Procedure
For photometric examination of the effect of different concentrations of NaCl the
erythrocytes will be placed in a series of decreasing hypotonic solutions.
Prepare and label 9 Eppendorff cups
Pipette according to the following scheme:
Cup Nr
0.155 M NaCl
A. dest.
Molarity
Absorption at
[µL]
[µL]
[M]
540 nm
1
0
1000
2
200
800
3
300
700
4
400
600
5
500
500
6
600
400
7
700
300
8
800
200
9
1000
0
Add 30 µL of erythrocyte suspension to each cup, mix carefully.
Incubate for 10 minutes.
Centrifuge for 5 minutes at 3000 rpm (rounds per minute)
Transfer the supernatant (~ 800 µL) into cuvettes – be sure not to touch or resuspend
the pellet!
Measure the absoption at 540 nm
Calculate the grade of haemolysis by setting the total haemolysis = 100%. Prepare a
diagram and interpret!
Restriction digest and DNA fingerprint

For every pipetting use a new tip!

Per tube you find 4µl of DNA (suspect 1 – 5 and crime scene, altogether six
samples).

Pipet 10µl of enzyme mix (EcoRI and PstI) and 4µl buffer into each DNA
sample. Use a new tip for each pipetting step.

Fill to 50µl with a. dest.

Close the tubes and mix by tapping on the tubes with a finger.

Centrifuge briefly to collect liquid at the bottom of the tube.

Restriction digest:
Incubate 45-60min at 37°C in a heating block.

During the digest each group prepares an own gel for the gelelectrophoresis.
o Mask gel trays accurately with tesa tape according to instructions of
your tutor!
o The gel consists of 0,7% agarose.
o For a small gel you have to calculate the amount of agarose for 50ml.
o Weigh the amount of agarose and fill with 50ml TBE buffer.
o Cook agarose in the microwave until it is boiling. Wear safety goggles!
o Add 5µl ethidium bromide according to the instructions of your tutor.
o After cooling down a bit you can cast the agarose in the chamber (make
sure that it is not too hot!!!).

After the digest chill your tubes on ice.

Centrifuge tubes briefly.

Pipet 17µl of each sample plus 3µl loading dye in a new tube.

Close tubes and mix briefly with a finger as before.

Put chilled agarose trays in the gel chamber and fill with buffer until gels are
covered.

Load the gel by pipetting 20µl of each sample into the slots. Note the order of
the samples!

Let gels run 1h at 100V..

After gelelectrophoresis evaluate and discuss your results!
Dr. Barbara Roitzheim
Department of
Natural Sciences
Hochschule
Bonn-Rhein-Sieg
University
of Applied Sciences
Practical Course for forign Master student
in Molecular Genetics
WS 2010/11
http://www.bioer.com.cn/en/shiji_lixinzhuduanhuahua.htm
Scientific Tutorial:
Dr. rer. nat. Andreas Pansky
Topics:
3. Isolation of Plasmid DNA using QIAprep Spin Miniprep
Kit
4. Determination and Characterisation of Plasmid DNA
-1-
Contents
3.
Isolation of Plasmid DNA .................................................................................. 3
3.1.
Isolation of Plasmid DNA using QIAprep Spin Miniprep Kit.......................... 3
3.1.1.
Principle ............................................................................................... 3
3.1.2.
Background information ....................................................................... 4
3.1.2.1.
Preparation and clearing of bacterial lysate ................................... 4
3.1.2.2.
DNA adsorption.............................................................................. 4
3.1.2.3.
Washing and elution of plasmid DNA............................................. 4
3.1.2.4.
DNA yield ....................................................................................... 4
3.1.2.5.
LyseBlue reagent ........................................................................... 5
3.1.2.6.
Growth of bacterial cultures ........................................................... 6
3.1.2.7.
Plasmid copy number..................................................................... 6
3.1.2.8.
Host strains .................................................................................... 6
3.1.2.9.
Inoculation...................................................................................... 6
3.1.2.10.
Antibiotics....................................................................................... 6
3.1.2.11.
Culture media................................................................................. 7
3.1.2.12.
Cell Lysates ................................................................................... 7
3.1.3.
4.
Protocol for QIAprep Spin Miniprep Kit ................................................ 8
3.1.3.1.
Material and Notes ......................................................................... 8
3.1.3.2.
Procedure ...................................................................................... 9
Determination and Characterisation of Plasmid DNA .................................. 10
4.1.
Determination of DNA Concentration and Ratio ......................................... 10
4.1.1.
Important note before starting ............................................................ 10
4.1.2.
Protocol ............................................................................................... 10
4.2.
Characterisation of DNA on an Agarose Gel .......................................... 12
4.2.1.
Digestion of the Plasmid DNA with Endonuncleases .......................... 12
4.2.2.
Agarose Gel Electrophoresis.............................................................. 12
X. Appendix .......................................................................................................... 13
X.1.
Solutions .................................................................................................... 13
X.2.
Definitions and Abbreviations ..................................................................... 13
X.3.
References ................................................................................................. 14
-2-
3.
Isolation of Plasmid DNA
The aim of this experiment is to isolate and purify plasmid DNA from an E.coli culture
by using the QIAprep Spin Miniprep Kit from the company QIAGEN.
To determine and characterise the isolated plasmid DNA the amount of DNA will be
measured using a photometer, followed by an enzymatic digestion for subsequent
examination of the plasmids on an agarose gel.
All the technical informations and most part of the script based on the QIAprep
Miniprep Handbook 12/2006 from Qiagen (1).
3.1. Isolation of Plasmid DNA using QIAprep Spin Miniprep
Kit
The QIAprep Miniprep system provides a fast and simple plasmid miniprep method
for routine molecular biology laboratory applications and is also useful for molecular
biology practical courses. These Kits use silica membrane technology to eliminate
the cumbersome steps associated with loose resins or slurries. Plasmid DNA purified
with QIAprep Mini Kits is immediately ready for use and does not need phenol
extraction and ethanol precipitation. The resulting plasmid DNA is of high-quality and
could be used for further experiments such as restriction enzyme digestion,
sequencing, transfection, ligation and transformation, library screening, and in vitro
translation.
Figure 1 shows a picture of the QIAprep mini spin columns with the corresponding
collection tubes.
Fig. 1: QIAprep spin column plus collection tubes
3.1.1.
Principle
The QIAprep mini procedure is based on alkaline lysis of bacterial cells followed by
adsorption of DNA onto silica in the presence of high salt (2). The unique silica
membrane used in this Kits completely replaces glass or silica slurries for plasmid
minipreps and all steps are performed without the use of phenol, chloroform, CsCl
and alcohol precipitation.
The procedure consists of three basic steps:
-
preparation and clearing of bacterial lysate
adsorption of DNA onto the QIAprep membrane (column)
washing and elution of plasmid DNA
-3-
3.1.2.
Background information
3.1.2.1.
Preparation and clearing of bacterial lysate
The QIAprep miniprep procedure uses the modified alkaline lysis method of Birnboim
and Doly (3). Bacteria are lysed under alkaline conditions, and the lysate is
subsequently neutralized and adjusted to high-salt binding conditions in one step.
After lysate clearing, the sample is ready for purification on the QIAprep silica
membrane. In the QIAprep Spin miniprep procedures, lysates are cleared by
centrifugation.
3.1.2.2.
DNA adsorption
QIAprep columns and plates use a silica membrane for selective adsorption of
plasmid DNA in high-salt buffer and elution in low-salt buffer. The optimized buffers in
the lysis procedure, combined with the unique silica 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.
3.1.2.3.
Washing and elution of plasmid DNA
Endonucleases are efficiently removed by a brief wash step with Buffer PB. This step
is essential when working with endA+ strains such as the JM series, HB101 and its
derivatives, or any wild-type strain, to ensure that plasmid DNA is not degraded. The
Buffer PB wash step is also necessary when purifying low-copy plasmids, where
large culture volumes are used. Salts are efficiently removed by a brief wash step
with Buffer PE. High-quality plasmid DNA is then eluted from the QIAprep column
with 50–100 µl of Buffer EB or water. The purified DNA is ready for immediate use in
a range of applications — no need to precipitate, concentrate, or desalt.
Note: Elution efficiency is dependent on pH. The maximum elution efficiency is
achieved between pH 7.0 and 8.5. When using water for elution, make sure that the
pH value is within this range. Store DNA at –20°C when eluted with water since DNA
may degrade in the absence of a buffering agent.
3.1.2.4.
DNA yield
Plasmid yield with the QIAprep miniprep system varies depending on plasmid copy
number per cell, the individual insert in a plasmid, factors that affect growth of the
bacterial culture, the elution volume (Figure 2), and the elution incubation time
(Figure 3). A 1.5 ml overnight culture can yield from 5 to 15 µg of plasmid DNA. To
obtain the optimum combination of DNA quality, yield, and concentration, we
recommend using Luria-Bertani (LB) medium for growth of cultures, eluting plasmid
DNA in a volume of 50 - 100 µl, and performing a short incubation after addition of
the elution buffer.
-4-
Fig. 2: 10 µg pUC18 DNA was purified using
the QIAprep Spin protocol and eluted with the
indicated volumes of Buffer EB. The standard
protocol uses 50 µl Buffer EB for elution, since
this combines high yield with high concentration.
However the yield can be increased by increasing
the elution volume.
Fig. 3: 10 µg pBluescript DNA was purified
using the QIAprep Spin Miniprep protocol and
eluted after the indicated incubation times with
either 50 µl or 100 µl Buffer EB. The graph
shows that an incubation time of 1 minute and
doubling the elution buffer volume increases
yield.
3.1.2.5.
LyseBlue reagent
Using a simple visual identification system, LyseBlue reagent prevents common
handling errors that lead to inefficient cell lysis and incomplete precipitation of SDS,
cell debris, and genomic DNA. LyseBlue can be added to the resuspension buffer
(Buffer P1) bottle before use. Alternatively, smaller amounts of LyseBlue can be
added to aliquots of Buffer P1, enabling single plasmid preparations incorporating
visual lysis control to be performed. LyseBlue reagent should be added to Buffer P1
at a ratio of 1:1000 to achieve the required working concentration (e.g., 10 µl
LyseBlue into 10 ml Buffer P1). Make sufficient LyseBlue/Buffer P1 working solution
for the number of plasmid preps being performed. LyseBlue precipitates after addition
into Buffer P1. This precipitate will completely dissolve after addition of Buffer P2.
Shake Buffer P1 before use to resuspend LyseBlue particles. The plasmid
preparation procedure is performed as usual. After addition of Buffer P2 to Buffer P1,
the color of the suspension changes to blue. Mixing should result in a
homogeneously colored suspension. If the suspension contains localized regions of
colorless solution or if brownish cell clumps are still visible, continue mixing the
solution until a homogeneously colored suspension is achieved.
Upon addition of neutralization buffer (Buffer N3), LyseBlue turns colorless. The
presence of a homogeneous solution with no traces of blue indicates that SDS from
the lysis buffer has been effectively precipitated (Figure 4.).
Fig. 4: Upon addition of lysis buffer the cell suspension turns blue allowing easy visualization of
aggregated cells. A Insufficient mixing after addition of lysis buffer. B Correct mixing after addition of
lysis buffer. Addition of neutralization buffer causes LyseBlue to turn colorless. A homogeneous
solution with no traces of blue indicates that the SDS has been effectively precipitated. C Insufficient
mixing after addition of neutralization buffer. D Correct mixing after addition of neutralization buffer.
-5-
3.1.2.6.
Growth of bacterial cultures
Plasmids are generally prepared from bacterial cultures grown in the presence of a
selective agent such as an antibiotic (4, 5). The yield and quality of plasmid DNA may
depend on factors such as plasmid copy number, host strain, inoculation, antibiotic,
and type of culture medium.
3.1.2.7.
Plasmid copy number
Plasmids vary widely in their copy number per cell, depending on their origin of
replication (e.g., pMB1, ColE1, or pSC101) which determines whether they are under
relaxed or stringent control; and depending on the size of the plasmid and its
associated insert. Some plasmids, such as the pUC series and derivatives, have
mutations which allow them to reach very high copy numbers within the bacterial cell.
Plasmids based on pBR322 and cosmids are generally present in lower copy
numbers.
3.1.2.8.
Host strains
Most E. coli strains can be used successfully to isolate plasmid DNA, although the
strain used to propagate a plasmid has an effect on the quality of the purified DNA.
Host strains such as DH1, DH5α, and C600 give high-quality DNA. The slower
growing strain XL1-Blue also yields DNA of very high-quality which works extremely
well for sequencing. Strain HB101 and its derivatives, such as TG1 and the JM
series, produce large amounts of carbohydrates, which are released during lysis and
can inhibit enzyme activities if not completely removed (4). In addition, these strains
have high levels of endonuclease activity which can reduce DNA quality. The
methylation and growth characteristics of the strain should also be taken into account
when selecting a host strain. XL1-Blue and DH5α are highly recommended for
reproducible and reliable results.
3.1.2.9.
Inoculation
Bacterial cultures for plasmid preparation should always be grown from a single
colony picked from a freshly streaked selective plate. Subculturing directly from
glycerol stocks, agar stabs, and liquid cultures may lead to uneven plasmid yield or
loss of the plasmid. Inoculation from plates that have been stored for a long time may
also lead to loss or mutation of the plasmid. The desired clone should be streaked
from a glycerol stock onto a freshly prepared agar plate containing the appropriate
selective agent so that single colonies can be isolated. This procedure should then
be repeated to ensure that a single colony of an antibiotic resistant clone can be
picked. A single colony should be inoculated into 1–5 ml of media containing the
appropriate selective agent, and grown with vigorous shaking for 12–16 hours.
Growth for more than 16 hours is not recommended since cells begin to lyse and
plasmid yields may be reduced.
3.1.2.10.
Antibiotics
Antibiotic selection should be applied at all stages of growth. Many plasmids in use
today do not contain the par locus which ensures that the plasmids segregate equally
during cell division. Daughter cells that do not receive plasmids will replicate much
faster than plasmid-containing cells in the absence of selective pressure, and can
quickly take over the culture. The stability of the selective agent should also be taken
into account. Resistance to ampicillin, for example, is mediated by β-lactamase which
is encoded by the plasmid linked bla gene and which hydrolyzes ampicillin. Levels of
ampicillin in the culture medium are thus continually depleted. This phenomenon is
-6-
clearly demonstrated on ampicillin plates, where “satellite colonies” appear as the
ampicillin is hydrolyzed in the vicinity of a growing colony. Ampicillin is also very
sensitive to temperature, and when in solution should be stored frozen in single-use
aliquots. The recommendations given in Table 1 are based on these considerations.
Table 1. Concentrations of Commonly Used Antibiotics
Antibiotic
Concentration
Storage
Ampicillin (sodium 50 mg/ml in water
salt)
Chloramphenicol
34
mg/ml
in
ethanol
Kanamycin
10 mg/ml in water
Streptomycin
10 mg/ml in water
Tetracycline HCl
5 mg/ml in ethanol
–20°C
Working
solution
(dilution)
100 µg/ml (1/500)
–20°C
170 µg/ml (1/200)
–20°C
–20°C
–20°C
50 µg/ml (1/200)
50 µg/ml (1/200)
50 µg/ml (1/100)
3.1.2.11.
Culture media
Luria-Bertani (LB) broth is the recommended culture medium for use with QIAprep
Kits, since richer broths such as TB (Terrific Broth) or 2x YT lead to extremely high
cell densities, which can overload the purification system. It should be noted that
cultures grown in TB may yield 2–5 times the number of cells compared to cultures
grown in LB broth. If these media are used, recommended culture volumes must be
reduced to match the capacity of the QIAprep membrane. If excess culture volume is
used, alkaline lysis will be inefficient, the QIAprep membrane will be overloaded, and
the performance of the system will be unsatisfactory. Furthermore, the excessive
viscosity of the lysate will require vigorous mixing, which may result in shearing of
bacterial genomic DNA and contamination of the plasmid DNA. Care must also be
taken if strains are used which grow unusually fast or to very high cell densities. In
such cases, doubling the volumes of Buffers P1, P2, and N3 may be beneficial. It is
best to calculate culture cell density and adjust the volume accordingly. Please note
that a number of slightly different LB culture broths, containing different
concentrations of NaCl, are in common use. Although different LB broths produce
similar cell densities after overnight culture, plasmid yields can vary significantly.
3.1.2.12.
Cell Lysates
Bacteria are lysed under alkaline conditions. After harvesting and resuspension, the
bacterial cells are lysed in NaOH/SDS (Buffer P2) in the presence of RNase A (3, 6).
SDS solubilizes the phospholipid and protein components of the cell membrane,
leading to lysis and release of the cell contents while the alkaline conditions denature
the chromosomal and plasmid DNAs, as well as proteins. The optimized lysis time
allows maximum release of plasmid DNA without release of chromosomal DNA, while
minimizing the exposure of the plasmid to denaturing conditions. Long exposure to
alkaline conditions may cause the plasmid to become irreversibly denatured (3). This
denatured form of the plasmid runs faster on agarose gels and is resistant to
restriction enzyme digestion. The lysate is neutralized and adjusted to high-salt
binding conditions in one step by the addition of Buffer N3. The high salt
concentration causes denatured proteins, chromosomal DNA, cellular debris, and
SDS to precipitate, while the smaller plasmid DNA renatures correctly and stays in
solution. It is important that the solution is thoroughly and gently mixed to ensure
complete precipitation. To prevent contamination of plasmid DNA with chromosomal
DNA, vigorous stirring and vortexing must be avoided during lysis. Separation of
-7-
plasmid from chromosomal DNA is based on co-precipitation of the cell wall-bound
chromosomal DNA with insoluble complexes containing salt, detergent, and protein.
Plasmid DNA remains in the clear supernatant. Vigorous treatment during the lysis
procedure will shear the bacterial chromosome, leaving free chromosomal DNA
fragments in the supernatant. Since chromosomal fragments are chemically
indistinguishable from plasmid DNA under the conditions used, the two species will
not be separated on QIAprep membrane and will elute under the same low-salt
conditions. Mixing during the lysis procedure must therefore be carried out by slow,
gentle inversion of the tube.
3.1.3.
Protocol for QIAprep Spin Miniprep Kit
3.1.3.1.
Material and Notes
QIAprep spin columns, Buffer P1, Buffer P2, Buffer N3, Buffer PB, Buffer PE, Buffer
EB, LyseBlue reagent, RNase A, LB-Medium, Antibiotics, 15 ml Falcon tubes, 1.5 ml
Eppendorf tubes.
Buffer notes
■ Add the provided RNase A solution to Buffer P1, mix, and store at 2–8°C.
■ Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume).
■ Check Buffers P2 and N3 before use for salt precipitation. Re-dissolve any
precipitate by warming to 37°C. Do not shake Buffer P2 vigorously.
■ Close the bottle containing Buffer P2 immediately after use to avoid acidification
of Buffer P2 from CO2 in the air.
■ Buffers P2, N3, and PB contain irritants. Wear gloves when handling these buffers.
■ Optional: Add the provided LyseBlue reagent to Buffer P1 and mix before use. Use
one vial LyseBlue (spin down briefly before use) per bottle of Buffer P1 to achieve a
1:1000 dilution. LyseBlue provides visual identification of optimum buffer mixing
thereby preventing the common handling errors that lead to inefficient cell lysis and
incomplete precipitation of SDS, genomic DNA, and cell debris.
Centrifugation notes
■ All centrifugation steps are carried out at 13,000 rpm in a conventional, table-top
micro-centrifuge.
Elution notes
■ Ensure that the elution buffer is dispensed directly onto the center of the QIAprep
membrane for optimal elution of DNA. Average eluate volume is ca. 48 µl from an
elution-buffer volume of 50 µl (QIAprep spin procedures) or ca. 95 µl from 100 µl
elution buffer.
■ For increased DNA yield, use a higher elution-buffer volume. For increased DNA
concentration, use a lower elution-buffer volume.
■ If water is used for elution, make sure that its pH is between 7.0 and 8.5. Elution
efficiency is dependent on pH and the maximum elution efficiency is achieved within
this range. A pH <7.0 can decrease yield. Note: Store DNA at –20°C when eluted
with water, as DNA may degrade in the absence of a buffering agent.
■ DNA can also be eluted in TE buffer (10 mM Tris·Cl, 1 mM EDTA, pH 8.0), but the
EDTA may inhibit subsequent enzymatic reactions.
-8-
3.1.3.2.
Procedure
1. Pick a single colony from a freshly streaked selective plate and inoculate a
culture of 1–5 ml LB medium containing the appropriate selective antibiotic.
Incubate for 12–16 h at 37°C with vigorous shaking.
Growth for more than 16 h is not recommended since cells begin to lyse and plasmid
yields may be reduced. Use a tube or flask with a volume of at least 4 times the
volume of the culture.
2. Harvest the bacterial cells by centrifugation at > 8000 rpm (6800 x g) in a
conventional, table-top microcentrifuge for 3 min at room temperature (15–
25°C).
The bacterial cells can also be harvested in 15 ml centrifuge tubes at 4700 x g for 10
min at 4°C. Remove all traces of supernatant by inverting the open centrifuge tube
until all medium has been drained.
3. Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a
microcentrifuge tube.
Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible
after resuspension of the pellet. If LyseBlue reagent has been added to Buffer P1,
vigorously shake the buffer bottle to ensure LyseBlue particles are completely
dissolved. The bacteria should be resuspended completely by vortexing or pipetting
up and down until no cell clumps remain.
4. Add 250 µl Buffer P2 and mix thoroughly by inverting the tube 4–6 times.
Mix gently by inverting the tube. Do not vortex, as this will result in shearing of
genomic DNA. If 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
min. If LyseBlue has been added to Buffer P1 the cell suspension will turn blue after
addition of Buffer P2. Mixing should result in a homogeneously colored suspension. If
the suspension contains localized colorless regions or if brownish cell clumps are still
visible, continue mixing the solution until a homogeneously colored suspension is
achieved.
5. Add 350 µl Buffer N3 and mix immediately and thoroughly by inverting the
tube 4–6 times.
To avoid localized precipitation, mix the solution thoroughly, immediately after
addition of Buffer N3. Large culture volumes (e.g. ≥5 ml) may require inverting up to
10 times. The solution should become cloudy. If LyseBlue reagent has been used,
the suspension should be mixed until all trace of blue has gone and the suspension
is colorless. A homogeneous colorless suspension indicates that the SDS has been
effectively precipitated.
6. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top
microcentrifuge.
A compact white pellet will form.
7. Apply the supernatants from step 4 to the QIAprep spin column by decanting
or pipetting.
8. Centrifuge for 60 s. Discard the flow-through.
9. Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB
and centrifuging for 60 s. Discard the flow-through.
This step is necessary to remove trace nuclease activity when using endA+ strains
such as the JM series, HB101 and its derivatives, or any wild-type strain, which have
high levels of nuclease activity or high carbohydrate content. Host strains such as
XL-1 Blue and DH5α™ do not require this additional wash step.
10. Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging
for 60 s.
-9-
11. Discard the flow-through, and centrifuge for an additional 2 min to remove
residual wash buffer.
Important: Residual wash buffer will not be completely removed unless the flowthrough is discarded before this additional centrifugation. Residual ethanol from
Buffer PE may inhibit subsequent enzymatic reactions.
12. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute
DNA, add 100 µl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each
QIAprep spin column, let stand for 3 min, and centrifuge for 2 min.
Note: maximum recommended culture volumes:
High-copy plasmids:
1.5 - 5 ml
expected yields:
Low-copy plasmids:
10 ml
expected yields:
4.
up to 20 µg DNA
5 – 10 µg DNA
Determination and Characterisation of Plasmid
DNA
To prove the quality of the isolated DNA, the DNA will be examined by two different
methods:
o
To determine the concentration and purity of the isolated plasmid DNA, the DNA
will be analysed and calculated in a photometer (GENEQUANT, AMERSHAM
BIOSCIENCE).
o
To determine the quality of the DNA and to prove the right size of the plasmid, it
will be separated on an agarose gel after restriction digest with appropriate
restriction enzymes2. The DNA in the ethidium bromide containing gel will be
visualized under UV light (320nm; ChemiDoc gel documentation system,
BIORAD).
4.1. Determination of DNA Concentration and Ratio
4.1.1.
Important note before starting
The quartz cuvettes are very expensive. Please handle with care!
4.1.2. Protocol

Dilute an aliquot of your isolated plasmid DNA (1/20 or 1/10) with water in a final
volume of 100 µl.

Pipet 70 µl of water as control (blank) into a microcuvette. Place the cuvette into
the photometer (check orientation) and press “set reference”.

After emptying the cuvette, pipet 70 µl of your diluted DNA containing sample into
the microcuvette, place it into the photometer, check orientation and press “DNA
measurement”.
- 10 -

When shown “instrument ready”, the printout will come automatically on the
printer. Label the slip with the name of the group and the name of the plasmid.

Suck off the sample and rinse the microcuvette with aqua dest.

The resulting printout of the GENEQUANT measurement gives you the
absorbance at 260, 280 nm, the ratios and the concentration of your genomic
DNA (Figure 5).

Check if the parameters have been set properly: the factor has to be 50 for DNA
(40 for RNA, 20 for oligos); the dilution has to be 10 or 20.
Fig. 5: Example of printout of the GENEQUANT photometer
The determination of purity of the DNA should result in a ratio (A260/A280; Fig. 4
asterix).
1,7 - 1,9 = DNA
<1,7 contamination with proteins or chemicals as phenol
>1,9 contamination with RNA
>2,0=RNA
The determination of the concentration will be acquired taking the A260nm (A260nm x
Factor of Dilution = µg/ml DNA; Fig. 4 star). This calculation is already done by the
gene quant photometer. The absorption at A260 has to be between 0.1 and 1.0 OD to
be in a reliable range (Figure 6). The resulting concentrations of the plasmid DNA will
be between 0.1 to 0.5 µg/µl (total up to 20 µg).
Fig. 6: Reliability of A260 readings for nucleic acid concentration determination
- 11 -
4.2. Characterisation of DNA on an Agarose Gel
After determination of DNA concentration, the plasmids will be digested with
appropriate restriction enzymes to control the plasmid and insert for proper size.
Restriction endonucleases of the multiple cloning site (MCS) are used. Their
restriction sites are unique in the plasmid, if the plasmid has no additional insert
(=gene of interest). In combination with the restriction site of the gene of interest, the
restriction pattern characterizes the plasmid3,4,5.
We will use several different enzymes to cut the isolated plasmid. Enzymes could be
HindIII, PstI, HaeIII, XbaI, BamHI, EcoRI and/or XhoI, respectively.
4.2.1. Digestion of the Plasmid DNA with Endonuncleases
Pipet the following substances into a 1.5 ml eppendorf tube for each of the provided
enzymes:
1 – 10 µl
y µl
2 µl
x µl
20 µl
plasmid DNA (0.5 – 1.0 µg)
restriction endonuclease (5 units total)
10 x restriction buffer
H2O up to achieve 20 µl final volume
Repeat the procedure for a second sample of each plasmid DNA without adding
restriction enzyme (control).
Incubate the restriction digest for a minimum of one to two hours at 37 °C.
4.2.2. Agarose Gel Electrophoresis
NOTE:
Wearing gloves is recommended while handling with ethidium bromide.
Wear protective shield while taking hot agarose out of microwave oven.

Prepare a 0.8% agarose gel in 1 x TBE (volume 100 ml)

Melt the agarose in a microwave oven, cool down to approx. 60-70°C, add 6 µl of
ethidium bromide stock solution (10 mg/ml), pour up the solution in a tray and add
a ten well comb. Wait until the gel is polymerised.

In the first slot load X µl of lambda Hind III DNA marker mixed with H2O and 6 x
sample loading dye.

Load 10 - 20µl of digested and undigested plasmid DNA samples, respectively,
with 2 - 4 µl sample loading dye each per well.

Run the gel electrophoresis with constant voltage (100 V) for one hour.

Visualize the restricted DNA under UV light (320nm) in the gel documentation
system.

Label the slip and analyse the length of the fragments compared to lambda
HindIII DNA marker or the lambda EcoRI/HindIII DNA marker.
- 12 -

Record your data.
1
2
3
4
5
M
bp
23,130
9,416
6,557
4,361
2,322
2,027
564
125
Fig. 7: Example of an 1,2 % agarose gel. The different bands reflect different restriction sites of the
insert. M:  Hind III DNA marker (8 fragments ranging in size from 125 to 23,130 bp as indicated), 1-4:
plasmid digested with different restriction enzymes, 5: plasmid undigested, asterix: plasmid band.
X.
Appendix
X.1. Solutions
LB (Luria-Bertani)-Medium
Bacto-tryptone
Bacto yeast extract
NaCl
Buffer P1
Resuspension buffer
Buffer P2
Lysis buffer
Buffer N3
Neutralization buffer
Buffer QBT
Equilibration buffer
Buffer PB
Wash buffer
Buffer EB
Tris-EDTA-buffer
10xTBE
Tris-borate-EDTA-buffer
10 g
5g
10 g, adjust pH to 7.0
50 mM Tris Cl pH 8.0
10 mM EDTA
100 µg/ml RNAse A
200 mM NaOH
1% SDS
3,0 M Potassium acetate pH 5.5
750 mM NaCl
50 mM MOPS pH7.0
15% isopropanol
0.15% Triton X-100
1,0 M NaCl
50 mM MOPS pH7.0
15% isopropanol
1 mM EDTA
10 mM Tris, pH 8.5
89 mM Tris pH 8.0
89 mM boric acid
8 mM EDTA
X.2. Definitions and Abbreviations
bp: base pairs
Colony: A visible clone of cells
- 13 -
Electrophoresis: A technique for separating the components of a mixture of
molecules (proteins, DNAs or RNAs) in an electric field within a gel.
Enzyme: A protein that functions as a catalyst.
Endonuclease: An enzyme that cleaves the phosphodiester bond within a nucleotide
chain.
Ethidium bromide: A molecule that can intercalate into DNA double helices when
the helix is under torsional stress.
Gene: The fundamental physical and functional unit of heredity, which carries
information from one generation to the next; a segment of DNA, composed of a
transcribed region and a regulatory sequence that make transcription possible.
Intercalating agent: Any molecule that inserts between two complementary base
pairs in a double-helical DNA or RNA molecule.
in vitro: In an experimental situation outside the organism (literally: “in glass”).
kb: Kilobase, 1000 nucleotide pairs.
Lysis: The rupture and death of a bacterial cell on the release of phage progeny.
Multiple cloning site: A vector DNA sequence containing multiple unique restrictionenzyme-cut sites, convenient for inserting foreign DNA.
Plasmid: Autonomously replicating extrachromosomal DNA molecule.
o
Low copy number plasmids: Lead to one replication per cell cycle.
o
High copy number plasmids: Due to the replication control mechanism
various replications per cell cycle, several copies of one plasmid per cell
(usually 10-20).
Plate: A flat dish used to culture microbes.
Restriction enzyme: An endonuclease that will recognize specific palindromic
nucleotide sequences in DNA and cuts the DNA chain at those points.
Restriction enzyme unit : A unit is defined as the amount of enzyme needed to
digest 1 µg of bacterial virus  DNA in 1 hour in a 50 µl reaction.
Star activity: A change in the recognition site specificity of certain restriction
endonucleases under suboptimal reaction conditions.
Vector: In cloning, the plasmid or phage chromosome used to carry the cloned DNA
segment.
X.3. References
1.
QIAprep Miniprep Handbook 12/2006 from Qiagen
2.
Vogelstein, B., and Gillespie, D. (1979) Preparative and analytical purification
of DNA from agarose. Proc. Natl. Acad. Sci. USA 76, 615–619.
3.
Birnboim, H.C. and Doly, J. (1979) A rapid alkaline lysis procedure for
screening recombinant plasmid DNA. Nucl. Acids Res. 7, 1513-1522.
4.
Sambrock, J. et al., eds. (1989) Molecular cloning : a laboratory manual, 2nd
ed., Cold Spring Harbor, Cold Spring Harbor Laboratory Press.
5.
Ausubel, F.M. et al., eds. (1991) Current protocols in Molecular Biology, New
York, Wiley Interscience
6.
Birnboim, H.C. (1983) A rapid alkaline extraction method for the isolation of
plasmid DNA. Methods Enzymol. 100, 243-255
7.
Griffiths, A.J.F. et al. (1999) Modern Genetic Analysis, New York, Freeman &
Co.
- 14 -
Magnetic activated cell sorting
Antibodies can be labelled with magnetic beads. There are several types of magnetic
beads on the market, mainly differing in size. We will use small paramagnetic beads
coupled to anti-CD4 antibodies to sort CD4+ T cells from human blood. These beads
are roughly of the size of a viral particle and do not disturb flow cytometric analysis or
biological experiments with the sorted cells.
The principle is very simple and allows sorting of several times 109 cells in a short
time to purities of up to 99%. Cells are stained with the antibodies and loaded on a
column containing small metal balls or steel wool. These columns are put into a
strong magnetic field for sorting. Magnetically labelled cells, those who bound the
antibody, will stick to the column. After washing off negative cells form the column,
the column is taken out of the magnetic field and cells are eluted from the column.
This way one can either positively enrich for cells sticking to the column or negatively
deplete cells bound to the column. The later one is very useful if the cells to be
enriched shall not be activated by bound antibodies. For this, one can use a mixture
of antibodies against every type of cell, except the one you are interested in.
Sample preparation
You get 1x 107 human leucocytes in a 1.5 mL Eppendorf tube
Fill up to 1 ml with MACS buffer
Centrifuge at 300 x g for 5 min at room temperature (RT)
Aspirate the supernatant
Resuspend pellet in 90 µL of MACS buffer
Magnetic labelling
Add 10 µL of CD4 Micro Beads
Mix by gently pipetting the cells up and down several times and incubate for 15 min in
a refrigerator. There cells should be completely resuspended, no clumps of cells left
over.
Add 20 µL of CD4-FITC antibody and incubate for 5 min in a refrigerator
Wash cells by adding 1mL of MACS buffer
Centrifuge at 300 x g for 10 min at 4°C
Aspirate the supernatant
Resuspend pellet in 500 µL of MACS buffer
Out of these 500 µL transfer 6 x 105 cells into a FACS tube (tube 1), add 200 µL
MACS buffer.
Place your FACS tube 1 on ice.
Magnetic separation
Take your MACS column out of the sterile package and insert into the magnet
Rinse column with 500 µL of MACS buffer  collect flow through (waste) into 1.5 mL
tube and discard it (the fluid will be turbid due to a protective coating of the column)
Wait till no fluid passes the column
Place the pre-separation filter (yellow plastics) on the MACS column. This is to avoid
clumps to enter the column
Now apply the cell suspension through the filter onto the column and start to collect
the flow through into a FACS tube (tube 2) placed on ice under the column.
Wash column 3 times with 500 µL of MACS buffer each, let the reservoir run empty in
between, collect total effluent as well in FACS tube 2
After the flow has stopped, take the column out of the magnetic field
Place column in a new FACS tube (tube 3).
Pipette 500 µL of MACS buffer onto the column
Immediately flush out by firmly applying the plunger
Collect effluent in the FACS tube 3
Evaluate tubes 1-3 with the FACS
Data analysis
Data will be analyzed using the Cell Quest Software
Graphs will be copy/pasted into PowerPoint for use in your protocol
You will analyze your 3 fractions (before the sort, flow through, sorted cells) and
quantify the purity of your sorted CD4 cells.
Figure 1: Principle of MACS sorting
Figure 2: FACS analysis of a typical successful CD4 sort. Black line, cells before separation, grey line
negative cells and lilac line positively sorted CD4 T cells.
Hochschule
Fachbereich
Bonn-Rhein-Sieg
Angewandte Naturwissenschaften
University of Applied Sciences
Department of Applied Sciences
Laboratory Course
in Instrumental Analysis
WS 2010 - 2011
Tutor:
Ute Eschweiler
Experiments:
SDS-PAGE of Proteins
Western Blot
© U. Eschweiler, Hochschule Bonn-Rhein- Sieg, 1. Modified Edition, 2010
Time schedule
1. Day
1. Introduction SDS PAGE
60 minutes
2. Casting and polymerization of separation gel
90 minutes
3. Sample preparation
30 minutes
4. Casting and polymerization of stacking gel
60 minutes
5. Storage of Gel
30 minutes
2. Day
1. Module Assembly and loading
60 minutes
2. Electrophoresis
30-45 minutes
3. Staining
60 minutes
4. Equilibration of Protein Gels
5. Preparation of Filter Paper and Nitrocelllulose Membrane
6 Stacking of the Western Blot
30 minutes
7 Western Blot run
35 minutes
Waiting time: Scanning of Gels
Cleaning, tidying up and refilling of equipment
8 Ponceau S Staining
15 minutes
9 Scanning of Gels and the Western Blot
30 minutes
September 2010
2
Contents
I Time schedule
1
Theoretical background SDS-PAGE ......................................................................4
2
Theoretical background Western Blot...................................................................5
3
Materials SDS -PAGE ..............................................................................................6
4
Materials Western-Blot ...........................................................................................7
5
Methods....................................................................................................................8
5.1 Gel Cassette Preparation...................................................................................................................... 8
5.2 Casting the Separating Gel (12% T) ..................................................................................................... 9
5.3 Casting the Stacking Gel (5% T)......................................................................................................... 10
6
Preparation of protein samples ...........................................................................11
6.1 Determination of Molecular Weight .................................................................................................... 11
6.2 Heating and Centrifugation ................................................................................................................. 11
7
Module Assembly ..................................................................................................12
8
Sample Application ...............................................................................................13
9
Running of gels .....................................................................................................13
10
Separation of the gel .............................................................................................14
11
Visualization ..........................................................................................................15
12
Western-Blot..........................................................................................................16
12.1 Transfer check (Ponceau S staining) ................................................................................................. 17
13
Final evaluation .....................................................................................................18
14
Appendix................................................................................................................19
September 2010
3
1 Theoretical background SDS-PAGE
SDS-PAGE stands for sodium dodecyl sulfate polyacrylamide gel electrophoresis. This
technique allows the separation of proteins in an electric field based on the molecular
weight (MW) only. Major applications are MW determination and quantification of proteins
as well as the determination of their purity. SDS-PAGE yields the total MW of monomeric
proteins. In case of oligomeric proteins, however, subunit MWs are detected instead.
Proteins are prepared for analysis by heat denaturation in the presence of a reducing
agent, such as -mercaptoethanol or dithiothreitol (DTT), and the anionic detergent SDS.
The reducing agent will break any disulfide linkages present in the protein. SDS will bind to
the peptide backbone fairly specifically in a mass ratio of about 1.4 g SDS per 1g protein.
By interacting with SDS, hydrogen bonds and other non-covalent interactions in the protein
are broken. After treatment with SDS and a reducing agent, the secondary and tertiary
structures of the protein are destroyed, so the peptide backbones are unfolded, and the
resulting SDS-protein-micelles are ellipsoidally shaped. Long peptide backbones (high
MW) will bind more charged SDS molecules than short ones (see above), so the proteinSDS complexes will ideally show a constant charge to mass ratio.
Electrophoresis of proteins will be conducted in a polyacrylamide gel matrix. Upon polymerization of acrylamide (monomer) and bisacrylamide (crosslinker) in the presence of ammonium persulfate (APS, initiator) and TEMED (activator), a three-dimensional polymer
network is formed. The pore size distribution of this gel can be controlled via the concentration of the crosslinker. More bisacrylamide results in a tighter network with smaller
pores; those gels can be used to analyze proteins with lower MW. The gel structure is thus
characterized by the total concentration of acrylamide and bisacrylamide (%T) and by the
ratio of bisacrylamide to (acrylamide + bisacrylamide) (%C).
In an electric field, charged analytes move according to their charge to mass ratios. In the
absence of a gel matrix, SDS derivatized proteins would move with identical speed due to
their constant charge to mass ratio. In a gel matrix, however, smaller proteins move at a
higher speed than larger proteins which are more restricted by the smaller pores of the gel.
The separation is thus based on a molecular sieve effect. A stacking gel with lower %T is
placed on top of the separation gel because it enhances the resolution of separation by
focusing the protein samples at the start. After separation, the proteins are visualized by
staining the gel with Coomassie, a blue dye, which specifically binds to hydrophobic and
basic sites of amino acids.
The Molecular Weight of unknown protein samples shall be determined.
To identify the approximate size of a molecule run on a gel electrophoresis a molecular
weight marker composed of different proteins of known size is used.
When used in gel electrophoresis, markers effectively provide a logarithmic scale by which
to estimate the size (i.e. the molecular weight) of the other fragments
By measuring the distance travelled by the markers and the protein of interest, one can
determine the approximate molecular weight of the experimental protein.
September 2010
4
2 Theoretical background Western Blot
The Western blot (alternatively, protein immunoblot) is an analytical technique used to
detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF,
where they are probed (detected) using antibodies specific to the target protein.
In order to make the proteins accessible to antibody detection, they are moved from within
the gel onto a membrane made of nitrocellulose or polyvinylidene difluoride (PVDF).
A method for transferring the proteins is called electroblotting and uses an electric current
to pull proteins from the gel into the PVDF or nitrocellulose membrane. The proteins move
from within the gel onto the membrane while maintaining the organization they had within
the gel. As a result of this "blotting" process, the proteins are exposed on a thin surface
layer for detection. Both varieties of membrane are chosen for their non-specific protein
binding properties (i.e. binds all proteins equally well). Protein binding is based upon hydrophobic interactions, as well as charged interactions between the membrane and protein. Nitrocellulose membranes are cheaper than PVDF, but are far more fragile and do
not stand up well to repeated probings.
The uniformity and overall effectiveness of transfer of protein from the gel to the membrane can be checked by staining the membrane with Coomassie Brilliant Blue or Ponceau S dyes. Ponceau S is the more common of the two, due to Ponceau S's higher sensitivity and its water solubility makes it easier to subsequently destain and probe the membrane as described below.
September 2010
5
3 Materials SDS -PAGE
Sample buffer:
63 mM Tris (pH 6.8), 2% (w/v) SDS, 5% (v/v) ß-Mercaptoethanol,
10% (v/v) Glycerol, 0.01% (v/v) Bromophenol blue
Electrode buffer:
25 mM Tris-HCl, 198 mM Glycine, 0.1% (w/v) SDS
Reagents:
Acrylamide / bisacrylamide solution: 30%T, 2.6 % C
A. dest.
1.5 M Tris-HCl, pH 8.8
1.0 M Tris-HCl, pH 6.8
10 % (w/v) SDS
10% (w/v) APS
TEMED
Marker:
PageRulerTM Unstained Protein Ladder (Fermentas)
Roti® -Mark Standard Protein Ladder (Roth)
Gel staining:
PageBlueTM Protein Staining Solution (Fermentas)
Instruments:
Mini-PROTEAN 3 Dodeca casting system (Biorad)
Samples:
BSA stock solution (0.8 mg/mL)
BSA samples of unknown concentration
Proteins of unknown MW
Other material:
Tert-amyl-alcohol
Apparatus and Setup
Tank and lid
Casting stand
Casting frame
Combs
Electrode assembly
Spacer plate
Short plate
September 2010
6
4
Materials Western-Blot
Buffer
Semidry-Transfer buffer
5.8 g Tris
2.9 g Glycine
0.38 g SDS
200 ml Methanol
Add ddH2O to 1000 ml.
Dye
Ponceau S
0.5 g Ponceau S
1 ml acetic acid
Add ddH2O to 100 ml, store at 4°C
Can be reused up to 5 times
Marker
PageRulerTM Prestained Protein Ladder (Fermentas
Instruments:
Whatman Fastblot B 34
Samples:
Proteins of unknown MW
Other material:
Nitrocellulose membrane
September 2010
7
5 Methods
Note: Acrylamide is a very strong neurotoxin. Use gloves at all times when ever you
handle the solutions.
PLEASE read the following instructions for preparing separation and stacking gels carefully before you start pipetting. Prepare appropriate pipettes with tips beforehand. Work
quickly since polymerization starts immediately after addition of APS and TEMED!
5.1 Gel Cassette Preparation
a) Clean plates with 70% denatured Ethanol. Place Short Plate on top of the Spacer
Plate. The orientation of the Spacer Plate should be such that the labelling is ‘up’.
b) Slide the two glass plates into the Casting Frame, keeping the short plate facing
the front of the frame; Cams should be in the open position, facing forward.
c)
Ensure that both plates are flush on a level surface. Leakage will occur if plates
are misaligned or oriented incorrectly.
d) When the glass plates are in place, engage the pressure cams to fasten the glass
cassette sandwich in the Casting Frame.
e) Place the Casting Frame into one of the positions in the Casting Stand by positioning the Casting Frame onto the casting gasket.
September 2010
8
5.2 Casting the Separating Gel (12% T)
a) The falcon tube provided by the tutor contains the following reagents:
Separation gel 12 %
6 mL
A. dest.
1.98 mL
30 % Acrylamide / bisacryl.
2.4 mL
1.5 M Tris-HCl (pH 8.8)
1.5 mL
10 % SDS
60 µL
To start polymerization of the gel, add 60 µL of APS and 6 µL of TEMED to the center
of the corresponding falcon tube, containing the Acrylamide / bisacrylamide solution.
Gently invert the falcon tube 3x (avoid formation of bubbles!!!) and immediately take up
the complete solution with a disposable syringe.
Note: Immediately continue with step c.
b) Pour the polymerizing solution in between the glass plates by allowing the solution to
flow down the spacer plate. Discontinue the addition once the solution reaches the
level 1 cm below the comb (upper base of the casting frame window). Once again:
avoid bubble formation!
If acrylamide mixture is left in the syringe (there should be some amount left!) pour it
back into the falcon tube and retain the tube for visual control of polymerization.
c) Carefully overlay the gel with a thin film (1-2 mm) of tert-Amyl alcohol (avoid disturbances of the interface) and allow to cast for 45 minutes.
d) Rinse the syringe (used for acrylamide) with A. dest (brown flask denoted by
“acrylamide waste”)!
September 2010
9
5.3 Casting the Stacking Gel (5% T)
The falcon tube provided by the tutor includes the following reagents:
Stacking gel 5 %
3 mL
A. dest.
2.04 mL
30 % Acrylamide / bisacryl.
0.51 mL
1.0 M Tris-HCl (pH 6.8)
0.39 mL
10 % (w/v) SDS
30 µL
a) Just before pouring the stacking gel: control if polymerization worked! If 'yes', decant
the film of tert-Amyl alcohol on a sheet of paper, rinse the surface twice with deionized
water and remove all free water with a small filter paper (do not touch the separation
gel surface with the filter paper).
b) In order to prepare the stacking gel, add 30 µL of APS and 3 µL of TEMED to the corresponding falcon tube, containing 3 ml Acryl/ Bisacrylamide solution. Gently invert the
falcon (avoid formation of bubbles) and immediately take up the complete solution with
a disposable syringe.
c) Pour the polymerizing solution on top of the separating gel. Discontinue the addition
once the solution reaches the level of the shorter glass plate. Insert a comb in between
the glass plates. To minimize bubble formation, insert the comb at an angle. Allow to
polymerize for 30 minutes.
d) Rinse the syringe (used for acrylamide) with A. dest (brown flask denoted by
“acrylamide waste”)!
September 2010
10
6 Preparation of protein samples
Note:
1. Before starting work centrifuge all defrosted samples at 10.000 rpm
(small table centrifuge) for 15 sec.
2. Preparing dilutions always start with the diluent!!
6.1 Determination of Molecular Weight
a) Two MW unknowns (A, B) are available in reaction tubes (5 µl each).
b) Add 10 µl of sample buffer to tube A and B.
c) The tube containing the Molecular Weight Marker is ready to use!
6.2 Heating and Centrifugation
a) Heat up all samples and the Marker for further denaturation, 4 minutes at 95°C.
Heat can be used to disrupt hydrogen bonds and non-polar hydrophobic interactions.
b) Centrifuge the samples and the marker at 10'000 x g for 5 minutes.
c) Use the supernatant for SDS-PAGE.
September 2010
11
7 Module Assembly
a) Carefully remove the cast gel from the casting stand
b) Place two gel sandwiches into the clamping frame with Short Plates facing inward.
Note: If an odd number of gels are to be run, use the buffer dam.
c) Lift the gel sandwiches or cassettes into place against the green gaskets. Make sure
the short plates sit just below the notch in the gasket.
d) While gently squeezing the gel sandwiches against the green gaskets, slide the arms
of the clamping frame over the gels, locking them into place. This forms the clamp
assembly.
e) Fill the inner chamber of the assembly with electrode buffer.
September 2010
12
8 Sample Application
a) Carefully pull out the comb and wash each pocket with electrode buffer.
b) Apply 10 µl of each sample but only 5 µl of marker into a pocket according
to table 2.
Lane
1
2
3
4
5
6
7
8
9
10
11
12
MW
unknown
BSA
sample
buffer
13
14
PreSample
sample
buffer
MW
unknown
Marker
MW
unknown
Staining
BSA
sample
buffer
sample
buffer
MW
unknown
stained
Marker
Western Blot
Table 2: Loading sequence of samples on gel
9 Running of gels
a) Place the electrode frame into the Dodeca separation chamber which can be used
to run up to 12 gels in parallel.
b) The chamber is filled with 2.5 L of electrode buffer, connected to a thermostat set to
10 °C and to a power supply set to 200 Volt.
c) Start run.
Note: Do not touch the set-up while the voltage is turned on
d) Run-time is to be controlled by the groups themselves.
d) Once the run is completed, turn off the power supply and remove the cables from
the separation chamber.
e) Take out the clamping frames and discard the electrode buffer into the sink.
September 2010
13
15
10 Separation of the gel
Note: Remember to wear gloves to handle the gel. The gel is now ready to be stained
or prepared for Western transfer.
a) Gently separate Spacer and Short plate from each other and lift off the Short plate.
The gel will stick to one of the two glass plates.
b) Use the Gel releaser to cut off the stacking gel. Dispose the stacking gel.
c) Mark the orientation of the separation gel by cutting the edge at the lower part
of lane 1.
d) Divide the gel into two halves, using the gel releaser
Stain one half with Coomassie Blue (see chapter 11)
Use the other half (containing the prestained Marker) for blotting the proteins
onto a nitrocellulose membrane (see chapter Western Blot).
September 2010
14
11 Visualization
Washing (green tank)
a) Add 100 mL of deionized water to the green tank and place the gel in.
b) Put the uncovered tank into a microwave oven and turn on high power for 1 min. Do
not boil.
c) Take out the tank from the microwave oven and continue washing with gentle agitation for 4 min.
d) Discard the wash.
e) Repeat 3 times.
Staining (blue tank)
a) Stain the gel in 20 mL PageBlueTM Protein Staining Solution.
b) Put the uncovered tank into the microwave oven and turn on high power for up to
30 seconds. Do not boil.
c) Take out the tank from the microwave oven and continue staining with gentle agitation for 20 min.
d) Keep dye in the box.
Note: Do not discard the dye after staining!
Washing (green tank)
a) Take the gel out of the staining solution.
b) Immerse the gel for 5 min in 100 mL of deionized water with gentle agitation.
c) Store the gel in deionized water.
Scanning
a) Place the gel on the scanner surface.
b) Remove air bubbles.
c) Run the scan program with the help of the tutor.
d) Take a copy on your USB stick.
September 2010
15
12 Western-Blot
Transfer of proteins onto a Nitrocellulose Membrane (Western Transfer).
Note: wear gloves and do not touch the membrane with your unprotected fingers.
a) Equilibrate the polyacrylamide gel in Semidry-Transfer buffer for 15 min on a
shaker. Gel equilibration time has to be consistent. Shortened times may affect
transfer efficiencies.
b) Cut two pieces of filter paper and one piece of the blotting membran to gel size.
c) Soak pieces of filter paper in Semidry-Transfer buffer.
d) Wet the Blotting Paper with Semidry-Transfer buffer.
Note: Use a ball-pen (biro) to label the membrane on the upper right side!
e) Place the wet membrane on top of a filter paper.
f) Remove air bubbles which might be included by carefully rolling out the blotsandwich with a plastic tube.
g) Place the polyacrylamide gel on top of the membrane.
h) Place the second soaked filter paper on top of the polyacrylamide gel.
i) Remove air bubbles which might be included by carefully rolling out the blotsandwich with a plastic tube.
j) Place the stack on the anode (+). The anode is the plate electrode on the body of
the blotting apparatus
k) Connect the lid with the body of the blotting apparatus. Take care that lid and body
are attached completely horizontally.
l) If you have assembled the “sandwich“for blotting, the set up should look as depicted
below.
Set up of filter paper, gel and
blotting membrane between the
plate electrodes. Blotting paper
and blotting membrane are
used in the same size as the
gel.
m) Calculate the required current necessary for an application at a constant current
of 2.5 mA/cm2 per gel.
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n) Switch on Power Supply, and allow proteins to be transferred onto the membrane
for 30 minutes.
o) Switch off the current.
p) Disconnect the Power Supply from the blotting apparatus.
q) Take off the lid.
r) Remove the blot-sandwich carefully and take the membrane out of the “sandwich”.
s) Mark the running direction of your membrane with a ball pen.
t) After every blotting procedure clean the plate electrodes with distilled water only.
u) A paper towel can be used for drying.
12.1 Transfer check (Ponceau S staining)
a) Stain the blotted membrane 5 to 10 minutes in 5 ml Ponceau S. Use the small blue
bowl.
b) Pour the stain back into the tube.
c) Destain the membrane with water until bands are visible.
d) Use a ball pen (no Edding!) to mark the bands of the Molecular Weight Marker.
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13 Final evaluation
a) Clearly label your scanned gel and Western Blot for the report (x-axis and y-axis).
b) Determine the MW of your unknowns as follows:
Measure the travel distance of each protein (from the beginning of the separation gel to
the final position).
Calculate the log MW from the MW values of the marker (given below).
Plot the log MW values of the marker (y-axis) versus the travel distance (x-axis) for the
calibration curve. Calculate a calibration function by linear regression.
Deduce the subunit MWs of the unknown proteins using the calibration function.
Fermentas Prestained Marker
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Fermentas Protein marker
18
14 Appendix
Good Pipetting Technique
1. Tip Filling and Consistency
When depressing and releasing the plunger, maintain a consistent rhythm, speed, and
technique.
Excessively fast aspiration can lead to splashing, aerosols, contamination of shaft and piston, and even volume loss of the sample.
2. Vertical Immersion Angle for Aspiration
Keep the immersion angle as close as possible to the vertical
position – otherwise the vertical liquid column is smaller and
too much sample will be aspirated.
(When dispensing the tip should be at a slight angle to the
vessel-wall in order to ensure good sample release.)
3. Tip Immersion Depth
Correct tip immersion depth is especially important for micro-volume pipettes. If the tip is
immersed too far, more liquid will be aspirated due to increased pressure. Liquid retained
on the tip surface can distort results. If the tip is not immersed far enough, air can be
drawn in, resulting in air bubbles and inaccurate volume.
Pipette Volume
Immersion Depth
2 and 10ul
1mm
20 and 100ul
2-3mm
200 and 1000ul
3-6mm
5000ul and 10ML
6-10mm
4. Dispensing Technique
For most applications, it is recommended to dispense with the end of the tip resting
against the vessel's wall. This reduces or eliminates sample remaining in the tip after dispensing. Remove the pipette by sliding the tip end up the side-wall in order to release any
remaining droplet at the tip orifice.
If dispensing directly into the liquid, reverse-mode pipetting is recommended in order to
prevent sample pick-up after dispensing.
Other useful links: http://www.bio-rad.com/cmc_upload/Literature/34668/4006191B.pdf
http://www3.bio-rad.com/cmc_upload/Literature/37363/4006158B.pdf
http://www.carl-roth.de/media/_en-de/usage/3029.pdf
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