E4xxx series - Consort bvba
Consort
MANUAL
E4100
E4200
E4300
E4400
E4500
November 2011
Table of contents
Warranty ................................................................ 1
Safety precautions..................................................... 2
General care and maintenance...................................... 3
Environmental conditions ............................................ 3
Fitting electrode cables .............................................. 4
Vertical gel electrophoresis .......................................... 4
Gel preparation ........................................................ 6
Gel selection ........................................................... 6
Gel pouring ............................................................. 8
Gel running ............................................................. 11
Solutions for SDS PAGE gels .......................................... 12
2-D electrophoresis.................................................... 13
Electroblotting ......................................................... 17
Buffer solutions for blotting ......................................... 19
Sequencing.............................................................. 20
Reagent preparation and gel volumes .............................. 21
Assembling the unit ................................................... 23
Preparation of samples for loading ................................. 23
Gel running and ending ............................................... 24
Solutions for DNA sequencing ........................................ 25
Denaturing gradient system.......................................... 26
Warranty
CONSORT guarantees that the unit you have received has been
thoroughly tested and meets its published specification.
This unit (excluding all accessories) is warranted against defective material and workmanship for a period of twelve (12) months
from the date of shipment ex factory.
CONSORT will repair all defective equipment returned during
the warranty period without charge, provided the equipment has
been used under normal laboratory conditions and in accordance
with the operating limitations and maintenance procedures outlined in this instruction manual and when not having been subject
to accident, alteration, misuse or abuse.
No liability is accepted for loss or damage arising from the incorrect use of this unit. CONSORT’s liability is limited to the repair
or replacement of the unit or refund of the purchase price, at
CONSORT’s option. CONSORT is not liable for any consequential
damages.
CONSORT reserves the right to alter the specification of its products without prior notice. This will enable us to implement developments as soon as they arise.
CONSORT products are for research use only.
A return authorisation must be obtained from CONSORT before
returning any product for warranty repair on a freight prepaid
basis.
E4xxx
1
Safety precautions
• When used correctly, these units pose no health risk.
• However, these units can deliver dangerous levels of electricity and are to be operated only by qualified personnel following the guidelines laid out in this manual.
• Anyone intending to use this equipment should read the complete manual thoroughly.
• The unit must never be used without the safety lid correctly
in position.
• The unit should not be used if there is any sign of damage to
the external tank or lid.
• Always isolate electrophoresis units from their power supply
before removing the safety cover. Isolate the power supply
from the mains first then disconnect the leads.
• Do not exceed the maximum operating voltage or current.
• Do not operate the electrophoresis units in metal trays.
• Acrylamide is a volatile, cumulative neurotoxin and suspected carcinogen. Wear effective protective clothing and follow
recommended handling and disposal procedures.
• Polymerised gels contain some unpolymerised monomer. Handle with gloves only. Following the replacement of a platinum
electrode have the unit inspected and approved by your
safety officer prior to use.
• Do not fill the unit with running buffer above the maximum
fill lines.
• Do not move the unit when it is running.
• Caution: during electrophoresis very low quantities of various
gases are produced at the electrodes. The type of gas produced depends on the composition of the buffer employed.
To disperse these gases make sure that the apparatus is run in
a well ventilated area.
2
E4xxx
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General care and maintenance
Units are best cleaned using warm water and a mild detergent. Water at temperatures above 60°C can cause damage
to the unit and components. The tank should be thoroughly
rinsed with warm water or distilled water to prevent build up
of salts but care should be taken not to damage the enclosed
electrode and vigorous cleaning is not necessary or advised.
Air drying is preferably before use.
The units should only be cleaned with the following: warm
water with a mild concentration of soap or other mild detergent (compatible detergents include dish washing liquid,
hexane and aliphatic hydrocarbons). The units should not be
left to in detergents for more than 30 minutes.
The units should never come into contact with the following cleaning agents, these will cause irreversible and
accumulative damage: acetone, phenol, chloroform, carbon
tetrachloride, methanol, ethanol, isopropyl alcohol alkalis.
In case of Rnase Decontamination clean the units with a mild
detergent as described above. Wash with 3% hydrogen peroxide (H2O2) for 10 minutes. Rinsed with 0.1% DEPC- (diethyl
pyrocarbonate) treated distilled water (Caution: DEPC is a
suspected carcinogen. Always take the necessary precautions
when using.) RNaseZAP™ (Ambion) can also be used. Please
consult the instructions for use with acrylic gel tanks.
Environmental conditions
This apparatus is intended for indoor use only.
This apparatus can be operated safely at an altitude up to
2000 m.
The normal operating temperature range is between 4°C and
65°C.
Maximum relative humidity 80 % for temperatures up to 31°C
decreasing linearly to 50 % relative humidity at 40°C.
The apparatus is rated Pollution Degree 2 in accordance
with IEC 664. Pollution Degree 2 states that: “Normally only
non-conductive pollution occurs. Occasionally, however, a
temporary conductivity caused by condensation must be expected”.
E4xxx
3
Fitting electrode cables
1. Note the position of the lid on the unit. This shows the correct polarity and the correct orientation of the cables, black
is negative and red positive.
2. Remove the lid from the unit. Note if the lid is not removed,
fitting the cables may result in un-tightening of the gold plug
and damage to the electrode.
3. Screw the cables into the tapped holes as fully as possible so
that there is no gap between the lid and the leading edge of
the cable fitting.
4.. Refit the lid.
Vertical gel electrophoresis
1. Clean a set of glass plates for each gel first with distilled
water and then with 70 % ethanol. One set of glass plates
constitutes one notched glass plate and one plain glass plate
with bonded spacers. When using a triple glass plate sandwich, two notched glass plates are required, one set of free
spacers and a set of plain glass plates with bonded spacers.
The plain glass plate is positioned outermost, then a notched
glass plate, free spacers and second notched glass plate. Alternatively, accessory notched glass plates with bonded spacers are available. All glass plates, modules and casting base
accessories must be completely dry during set–up. Wet
components are more likely to miss-align and cause leaks.
2. Assemble the glass plates so that the bottom of the glass
plates and the spacers are perfectly aligned. For triple plate
sandwiches, the free spacers need to be perfectly aligned
which is best performed using a small spacer or comb to push
the spacers apart. Notched glass plates with bonded spacers
do not need manual alignment. Note: the glass plates with
bonded spacers have an arrow in the top of the spacers
which are slightly longer than the glass plate to indicate
the top.
3. The slab gel insert contains pressure bars which impart even
pressure onto the glass plates and allow even screw pressure
transfer onto the sealing edge of the glass plate, ensuring
complete sealing. Ensure that the pressure bars are adequately open for the thickness of spacer used. The bar can
be opened by loosening the screws or by sliding the clamps.
When using a triple glass plate sandwich, the pressure bars
will need to be in the completely open position.
4
E4xxx
4. Position the slab gel insert on a flat surface. Do not at this
stage insert the slab gel insert into the casting base.
5. Insert the glass plates into the Slab Gel Insert between the
pressure bar and the blue gasket and fully tighten the pressure bar screws in the order top then bottom. Fully tighten
the screw for the mini vertical and the screws sequentially
and in an even manner for the maxi vertical in the order middle two, top then bottom, making sure not to wobble the
unit. When only one gel is being run, the dummy plate must
be used in the second position and fully tightened. At this
stage, check that the bottom edges of the spacers and glass
plates are perfectly aligned.
6. Position the slab gel insert in the casting base such that the
cam pins have handles pointing downwards and are located in
the insert holes. The top of the running module may need to
be pushed down very slightly to locate the cam pins. With the
cam pin handles facing directly downwards, turn the cam pins
fully through 180° or until the insert has tightened onto the
silicone mat. It is best to turn the cams in opposite directions to each other. Do not overturn as this will cause the
glass plates to push upwards and the assembly will be more
likely to leak. The unit is now ready for gel preparation and
pouring.
Note: always reverse the silicone mat after casting to avoid
indentations from persisting. Never leave the casting upstand with glass plates tightened into the casting base for
long periods of time as this will also cause indentations in the
silicone mat.
E4xxx
5
Gel preparation
1. It is always advisable to work with pre-made stock solutions
which allow added convenience and save time when it comes
to gel pouring. The protocol below is given for use of the
standard stock solutions advised. This should be adjusted if
you are using different stock solutions or gel formulas.
2. Table below shows the volume of agarose solution required to
make the desired gel (1 mm thick) for each unit size. Adjustments are needed for 0.75, 1.5 or 2 mm spacers.
Model
Gel size (cm)
Volume (ml)
E4100
10x10 (one gel + dummy plate)
7.5
10x10 (two gels)
15
10x10 (four gels +triple plates)
30
E4200
E4300
E4400
20x10 (one gel+ dummy plate)
17.5
20x10 (two gels)
35
20x10 (four gels +triple plates)
70
20x20 (one gel+ dummy plate)
35
20x20 (two gels)
70
20x20 (four gels +triple plates)
140
Gel selection
1. Care should be taken when selecting the pore size of the gel
to be used. The pore size or % of gel determines the resolving
ability given different sizes of protein. See table below which
details and which percentage of gel to use to separate the
sizes of proteins indicated.
Acrylamide percentage
Separating resolution
5%
60...220 kDa
7.5 %
30...120 kDa
10 %
20...75 kDa
12 %
17...65 kDa
15 %
15...45 kDa
17.5 %
12...30 kDa
2. Prepare gel solutions as per tables below. These give the volumes of solutions from the standard stock solutions. These
should be gently mixed avoiding generation of bubbles which
will inhibit polymerization by removing free radicals.
6
E4xxx
Preparation of the separating gel solution for 10x10 cm gels
using 1 mm spacers.
Solution
5%
7.5
%
10
%
12
%
distilled water
8.7
7.5
6.3
5.25 3.75 2.5
30 % stock acrylamide
2.5
3.75
5
6
4x resolving Tris
3.75
3.75
3.75 3.75 3.75 3.75
10 % ammonium
persulphate
0.15
0.15
0.15 0.15 0.15 0.15
ml
15
%
7.5
17.5
%
8.75
Preparation of the separating gel solution for 20x10 cm gels
using 1 mm spacers.
12
%
15
%
17.5
%
5%
7.5
%
10
%
distilled water
20.5
17.6
14.8 12.3 8.8
30 % stock acrylamide
5.8
8.8
11.7 14.1 17.6 20.5
4x resolving Tris
8.8
8.8
8.8
10 % ammonium
persulphate
0.35
0.35
0.35 0.35 0.35 0.35
Solution
ml
8.8
8.8
5.8
8.8
Preparation of the separating gel solution for 20x20 cm gels
using 1 mm spacers.
Solution
5%
7.5
%
10
%
distilled water
41
35.3
29.6 24.7 17.6 11.7
30 % stock acrylamide
11.7
17.6
23.5 28.2 35.3 41.1
4x resolving Tris
17.6
17.6
17.6 17.6 17.6 17.6
10 % ammonium
persulphate
0.70
0.70
0.70 0.70 0.70 0.70
ml
E4xxx
12
%
15
%
17.5
%
7
Gel pouring
For gels with stacking layers
1. Insert the comb into the glass plates and mark a point on the
glass plates 1 cm below where the comb teeth finish. This
indicates where to add the resolving gel to.
2. Add 15 µl of TEMED to the resolving gel solution for 10x10 cm
sized gels, 35 µl for 10x20 cm sized gels and 70 µl for 20x20
cm sized gels and mix well but avoid generating air bubbles.
3. Fill the glass plates again avoiding generating any air bubbles.
Filling must be performed quickly before the TEMED causes
the gel to become too viscous.
4. Overlay the gel extremely carefully with 1 ml of Isobutanol,
Isopropanol or distilled water. When using distilled water extra care must be taken to ensure there is no mixing with the
gel solution.
5. Let the resolving gel polymerize. Usually this takes around 15
minutes but this can vary due to the freshness of the reagents
used. If polymerization is taken a lot longer than this, use
fresher stock solutions or add more APS and TEMED.
6. Prepare the stacking gel using table below as a guide.
Solution
E4100
E4200
ml
E4300
E4400
distilled water
4.2
8.4
16.8
30 % stock acrylamide
0.65
1.3
2.6
4x resolving Tris
1.6
3.2
6.4
10 % ammonium persulphate
0.067
0.134
0.268
7. Carefully mix the stacking gel solution, avoiding generating
air bubbles.
8. Pour off the overlay liquid and rinse the gel with distilled
water.
9. Add 6.7 µl of TEMED to the stacking gel solution for 10x10 cm
gels, 13.4 µl for 10x20 cm gels add 26.8 µl for 20x20 cm gels.
Mix well. Use a Pasteur pipette to fill the glass plates up to
the top with stacking gel solution.
10. Carefully insert the comb making sure that no air bubbles get
trapped under the ends of the comb teeth as these will inhibit
sample progression.
11. Allow the stacking gel polymerize for 30 minutes.
8
E4xxx
For gels without stacking layers
1. Add 15 µl of TEMED to the resolving gel solution for 10x10 cm
sized gels, 35 µl for 10x20 cm sized gels and 70 µl for 20x20
cm sized gels and mix well but avoid generating air bubbles.
2. Fill the glass plates again avoiding generating any air bubbles.
Filling must be performed quickly before the TEMED causes
the gel to become too viscous.
3. Carefully insert the comb making sure that no air bubbles get
trapped under the ends of the comb teeth as these will inhibit
sample progression.
4. Let the resolving gel polymerize. Usually this takes around 15
minutes but this can vary due to the freshness of the reagents
used. If polymerization is taken a lot longer than this, use
fresher stock solutions or add more APS and TEMED.
Preparation of denaturated protein samples for loading
The instructions given below are for denatured samples. For native samples, please consult a laboratory handbook.
1. Prepare the protein samples for loading. The volume of sample depends on the capacity of the wells.
2. Using a 0.5 ml micro-centrifuge tube or other convenient receptacle, combine the protein sample and 4x sample buffer.
It is always advisable to use protein markers in one of the end
lanes to indicate sizes of bands. These should be prepared
according to the manufacturers instructions.
3. Heat the samples in a water bath or heating block for 2 minutes to denature the samples.
4. Centrifuge the samples in a micro-centrifuge for 20 seconds
at 12000 rpm. The protein samples are now ready to load.
E4xxx
9
Loading the samples
1. If desired, fit the cooling pack(s) into the end of the tank.
These should be pre-frozen and fitted with the longest side
positioned sideways with the end(s) of the tank and pressed
into the recess. Or these can be fitted down the front of the
tank. Never fit these underneath the module in the bottom
of the tank as this will prevent the flow of current through
the gel and cause slow runs and over-heating.
2. Transfer the Inner gel module containing cast gels into the
main tank in the correct orientation as indicated, [+ve] on the
module aligned with [+ve] on the tank, [-ve] on the module
aligned with [–ve] on the tank.
3. Fill the outer tank with 1 x reservoir buffer solution. Below
table shows the volume of buffer required.
Buffer volume
E4100
ml E4200
Minimum
250
Inner tank is filled to above the wells.
Outer tank is filled to just flood the bottom of the glass plates. Cooling potential 500
is at a minimum which may affect resolution.
Maximum
1200
Inner tank is filled to above the wells.
Outer tank is filled to the maximum fill
line. Cooling is high offering good resolu- 2800
tion of samples.
Using the cooling packs
1000
Inner tank is filled to above the wells.
Cooling packs are inserted behind the
gels.
Outer tank is filled to the maximum fill 2300
line. Cooling is at a maximum.
E4300
E4400
1200
1800
5600
8400
4600
6900
4. Load the samples into the wells using a pipette tip taking care
not to damage the wells or induce any air bubbles.
5. Fill any unused wells with 1 x sample buffer.
6. It is a good idea to note the orientation and order the samples
were loaded in. This can be done by noting which samples
were loaded adjacent to each electrode.
10
E4xxx
Gel running
1. Fit the lid and connect to a power supply.
2. Consult below table for details on recommended power supply voltage settings.
3. Turn the power supply off when the loading dye reaches the
bottom of the gel, sooner if your proteins are below 4 kDa in
size.
4. Remove the gel running module, first emptying the inner
buffer into the main tank. Buffer can be re-used but this may
affect run quality if continued.
5. Unscrew the glass plates and gently pry apart the glass plates.
The gel will usually stick to one of the plates and can be removed by first soaking in buffer and then gently lifting with a
spatula.
6. The gel is now ready to be stained with Coomassie or silver
stain or the proteins in the gel can be transferred to a membrane by electroblotting for specific band identification and
further analysis.
Recommended voltage and
resultant current for 1 mm
thick, 12 % gels.
E4100
E4200
E4300
E4400
One gel
90...225 V
120...250 V
20...45 mA
20...45 mA
Two gels
Three gels
90...225 V
120...250 V
40...90 mA
40...90 mA
90...225 V
120...250 V
60...135 mA 60...135 mA
Four gels
90...225 V
120...250 V
80...180 mA 80...180 mA
E4xxx
11
Solutions for SDS PAGE gels
Stock 30% acrylamide gel solution
• 30.0 g acrylamide
• 0.8 g methylene bisacrylamide
• add distilled water to a final volume of 100 ml
Stock 4 x resolving gel Tris (1.5 M Tris.HCl pH 8.8, 0.4 % SDS)
• to 110 ml distilled water add 36.4 g of Tris base
• add 8 ml of 10 % SDS
• adjust pH to 8.8 with 1 M HCl
• add distilled water to a final volume of 200 ml
Stock 4 x stacking Tris (0.5 M Tris.HCl pH 6.8, 0.4 % SDS)
• to 110 ml distilled water add 12.12 g of Tris base
• add 8 ml of 10 % SDS
• adjust pH to 6.8 with 1 M HCl
• add distilled water to a final volume of 200 ml
Stock 4 x Tris-glycine tank buffer - SDS
• 36 g Tris base
• 172.8 g glycine
• add distilled water to a final volume of 3000 ml
1 x Tris-glycine tank buffer - SDS
• 750 ml of 4 x Tris-glycine reservoir buffer - SDS
• 30 ml of 10 % SDS
• add distilled water to a final volume of 3000 ml
10 % AP (ammonium persulphate solution)
• 0.1 g ammonium persulphate
• 1 ml distilled water
TEMED
Stock 4 x sample buffer
• 4 ml glycerol
• 2 ml 2-mercaptoethanol
• 1.2 g SDS
• 5 ml 4 x stacking Tris
• 0.03 g bromophenol blue
• aliquot into 1.5 ml microcentrifuge tubes. Store at -20°C.
12
E4xxx
2-D electrophoresis
Capillary tube gel pouring
There are two methods which can be used for tube gel casting.
Method-1 details casting by injection, method-2 details casting
by capillary action.
Method-1: filling by injection
1. Place the appropriate number of capillary tubes into the Tube
Gel Running module, inserting these carefully from the top.
2. Seal the bottom ends of the tubes using NescoFilm.
3. Prepare the following solution:
• 16 ml distilled water (18 ml for native gels)
• 2.4 ml glycerol
• 0.9 ml 4-8 resolyte or other commercially available 40 %
ampholyte solution
• 3.8 ml acrylamide/bis solution
• 15 µl TEMED
• 16.2 g urea (omit for native gels)
• 0.6 ml NP40 (omit for native gels)
This will be enough to pour twenty 80 mm capillary tubes or
ten 170 mm capillary tubes. This solution should be de-gassed
prior to pouring. When ready to pour, add 120 µl of 10 % w/v
ammonium persulphate solution.
4. Using a Hamilton or similar syringe, insert the needle into
the tube and carefully inject the solution so that the tubes
fill from the bottom. Keep filling to 1 cm of the length of the
tubes. The tubes can be gently tapped to get rid of air bubbles.
5. Fill the remaining 1 cm gap with water saturated isobutanol.
6. Leave to fully polymerise, which will normally take 1...2
hours.
7. After polymerisation, remove the water-saturated isobutanol. Tube gels can be used immediately or stored wrapped in
a damp paper towel and Nescofilm at 4°C. The Nescofilm at
the bottom of the tubes must be removed prior to electrophoresis.
E4xxx
13
Method-2: filling by capillary action
1. Place the appropriate number of capillary tubes in a suitable
outer receptacle such as a 15 ml falcon tube.
2. The amount of acrylamide required depends on the size of
the outer receptacle used. The larger the outer receptacle
used, the more acrylamide wastage so the following advised
volumes may need to be increased.
3. Prepare the following solution:
• 32 ml distilled water (18 ml for native gels)
• 4.8 ml glycerol
• 1.8 ml 4-8 resolyte or other commercially available 40 %
ampholyte solution
• 7.6 ml acrylamide/bis solution
• 30 µl TEMED
• 32.4 g urea (omit for native gels)
• 1.2 ml NP40 (omit for native gels)
This will be enough to pour twenty 80 mm capillary tubes or
ten 170 mm capillary tubes. This solution should be de-gassed
prior to pouring. When ready to pour, add 240 µl of 10 % w/v
ammonium persulphate solution.
4. Allow the tubes to equilibrate for a few moments.
5. Check the height of the acrylamide in the tubes. If the tubes
are full so that there is less than a 1 cm non-filled space at
the top, remove some of the acrylamide solution from the
beaker until the height is 1 cm from the top. If there is a
greater than 1 cm space at the top, add more acrylamide
solution, so that the solution rises in the tubes until there is a
1 cm space at the top.
6. When the solution has reached to within 1 cm of the top of
the tube, stop adding the acrylamide solution.
7. Fill the remaining 1 cm gap with water saturated isobutanol.
8. Leave to fully polymerise, which will normally take 1...2
hours.
9. After polymerisation, remove the water-saturated isobutanol. Tube gels can be used immediately or stored wrapped in
a damp paper towel and Nescofilm at 4°C. The Nescofilm at
the bottom of the tubes must be removed prior to electrophoresis.
10. The tubes may contain a residual of acrylamide on the outside
and may need cleaning with distilled water before insertion
into the tube gel insert.
14
E4xxx
1st dimension (IEF) phase tube gel running
Buffer and run conditions will vary according to the type of ampholyte used. The following conditions are given as guidelines
only and apply when 4-8 Resolyte is the ampholyte used. Other
Ampholytes will require different buffer solutions. Please consult manufacturer’s instructions.
1. Prepare ca. 500 ml of 10 mM H3PO4 Anode Buffer (1 litre for
E4200, 2 litres for E4300) and use this to fill the bottom chamber of the unit so that the bottoms of the capillary tubes are
submerged. If less than 10 capillary tubes are to be run, block
up the unused tube slots in the internal running module with
the blanking plugs provided. For high resolution separations,
we recommend filling the lower chamber completely with
buffer and using a pre-frozen cooling pack(s).
2. Place the internal running module into the unit and fill the
upper buffer reservoir with ±100 ml of 20 mM NaOH cathode
buffer (200 ml for E4200, 400 ml for E4300) so that the tops
of the capillary tubes are submerged.
3. For the prefocus, load the gels with 10 µl of 1 % ampholyte
solution and run for 15 minutes at 200 V, then for 30 minutes
at 300 V and then finally 30 minutes at 400 V. The prefocus
stage is recommended as it helps set up the pH gradient.
4. Load the tubes with the samples. These should be dissolved in
1 % ampholyte with 20 % glycerol.
5. Replace the safety lid firmly making sure that the electrical
connectors form a good contact.
6. Connect the electrophoresis apparatus to the power pack and
connect the power pack to the mains supply. Turn all settings
to zero before turning on the mains supply.
7. Run at 400 V for 3 hours and then 800 V for 30 minutes. These
conditions are for 8 cm tubes. 17 cm tubes need to be run at
400 V for 18 hours and then 800 V for 1 hour.
8. At the end of the run, turn the power supply settings to zero,
turn off the mains supply and disconnect the power leads.
9. Remove the internal module and remove the tubes from their
slots. The gels can be extracted from the capillary tubes by:
• inserting a piece of wire with a small plug of cotton wool on
the end and using this as a piston to push the gel out.
• inserting a Gilson tip into the end of the gel and gently
squeezing the gel out with air or water. Whichever of these
two methods is used, the gels should be handled with care
as they are fragile.
E4xxx
15
2-D size determination phase
1. To prepare the tube gel(s) for the 2-D, size-determining
phase, equilibrate them by soaking for 30 minutes in the running buffer to be used for the 2-D phase.
2. Remove the gel(s) from the running buffer pre-soak, and
place each lengthways onto the top of a pre-poured slab gel.
The slab gel should be cast using a blank or 2-D comb. For
details on the casting of slab gels see the previous pages in
this manual.
3. Hold the tube gel in place by pouring over it a low % agarose
gel containing the tracker dye.
4. Electrophorese as usual for slab gels until the tracker dye has
advanced the required distance down the gel.
5. The samples can be visualized using any of the standard staining methods or can be blotted.
16
E4xxx
Electroblotting
Setting up the blot sandwich
1. Each blot sandwich should be set up as follows:
• Cassette clamp [-ve] (black) side placed in a tray or other
suitable surface.
• Pre-soaked fibre pad (note two can be used with thin gels).
• Two pieces of thick filter paper, about 2...3 mm thick, presoaked in buffer.
• Gel.
• Transfer membrane. Usually this requires pre-soaking but
consult the manufacturers instructions for the type of membrane you are using. This should be smoothed so that no air
bubbles have been trapped.
• Two pieces of thick filter paper, about 2...3 mm thick, presoaked in buffer.
• Pre-soaked fibre pad (note two can be used with thin gels).
• Cassette clamp [+ve] (red) side slotted into the groove in
the bottom of the black cassette.
2. Close the hinge carefully so as to not disturb the sandwich.
3. Fill the tank with buffer solution up to the maximum fill line
indicated on the side of each unit. Improved transfer can usually be obtained by using chilled buffer.
Buffer volume
E4100
E4200
E4300
One cassette
1380
2800
5600
Two cassettes
1290
2620
5240
Three cassettes
1200
2440
4880
Four cassettes
1110
2260
4520
ml
Each cooling pack will take the place of 100 ml of buffer for
E4100 and 500 ml of buffer for E4200 and E4300.
E4xxx
17
Blot run conditions
1. Insert the cassettes into the slots in the module with the black
side of each adjacent to the negative electrode. It is a good
idea to note the orientation and order the blot sandwiches
were loaded in. This can be done by noting which samples
were loaded adjacent to each electrode.
2. Use of a magnetic stirring bar and plate is recommended to
mix the buffer to give consistency of transfer. A 4 mm diameter stirring bar should be placed underneath the module,
in the centre of the tank. The cooling pack provided, prefrozen, can be inserted at the side or front of the tank for
extended blots. Additional cooling packs can be purchased as
accessories to further aid cooling.
3. Insert the module, fit the lid and connect to a power supply.
4. Consult below table for details on recommended power supply voltage settings and blot times. Please note voltage and
current will vary according to the amount of cassettes, type
and temperature of buffer and thickness and percentage of
gel. This will also affect quality of transfer so time course of
transfer should be performed for your particular samples and
conditions.
5. When the blot time is completed, turn the power supply off.
6. Remove the cassettes from the main tank. Buffer can be reused but this may affect run quality if continued.
7. Lift the hinge of each cassette and gently pry apart the blot
sandwich and remove the membrane from the gel.
8. The membrane is now ready to be probed.
Duration of blotting
E4100
E4200
E4300
One hour
100 V
100 V
400 mA
400 mA
50 V
50 V
200 mA
200 mA
Three hours
18
E4xxx
Buffer solutions for blotting
Towbin Buffer
• 25 mM Tris,
• 192 mM glycine,
• 20 % methanol pH 8.3
Towbin Buffer SDS
• 25 mM Tris
• 192 mM glycine
• 20 % methanol pH 8.3
• 0.05-0.1 % (w/v) SDS
Bjerrum and Schafer-Nielsen Buffer
• 48 mM Tris
• 39 mM glycine
• 20 % methanol pH 9.2
Dunn Buffer
• 10 mM NaHCO3
• 3 mM NaCO3
• 20 % methanol pH 9.9
Do not adjust the pH when making these buffers as this will
cause blot over-heating. The pH will vary according to the
freshness of the reagents used.
E4xxx
19
Sequencing
Cleaning and preparation of glass plates
1. Better and more consistent results will be obtained if care is
taken to ensure that the glass plates are as clean as possible.
New glass plates must be cleaned in the same way as used
glass plates because these will contain surface debris that
may interfere with the gel.
2. First, clean using a neutral detergent and a small brush. Do
not use metal wool or other test tube brushes, abrasive cleaning creams or scourers because these can scratch the surface
of the glass plates.
3. The glass plates should be washed in the following sequence:
distilled water ethanol, acetone, ethanol, distilled water.
Thoroughly rinse and dry the glass plates before use. For extra clean plates, these should be wiped with a microscope tissue soaked in chloroform or dichloroethane in a fume hood.
4. To ease separation of the gel from the glass plates once the
gel has been run, it is advisable to siliconise the notched glass
plate with a tissue soaked in dimethyldichlorosilane. Wipe
the plate, including the ears, in a fume hood. Rinse with water and dry with a tissue.
5. The plain glass plate should be siliconised along the outer 1
cm lengths where the spacers will be positioned. This should
be periodically repeated when the gels start to stick to the
plates. Plates should then be cleaned and siliconised again.
6. The horizontal gel pouring method described in this manual
will not work if both plates are siliconised. In that case, use
an alternative gel pouring method or do not siliconise the
plain glass plate as described above.
7. The above procedures are not necessary every time a gel is
poured.
8. After use, first, clean using a neutral detergent and a small
brush and wahs with distilled water, ethanol and acetone as
described above.
1.
2.
3.
4.
5.
Reagent preparation and gel volumes
For consistent gels it is advisable to use high quality reagents
and where possible deionisine, degass and filtrate acrylamide
gel solutions prior to use.
Made up acrylamide solutions should be stored in a refrigerator and allowed to reach room temperature prior to pouring.
It is always advisable to work using stock solutions which
allow added convenience and save time when it comes to
gel pouring. Pages 12 list stock solutions for SDS PAGE gels
which should be pre-made beforehand. For DNA sequencing
see page 18. For native gel formulae and running conditions,
please consult a laboratory manual.
As a guide, polymerisation conditions should be adjusted
to effect polymerisation within about 5...15 minutes. Test
a small volume in a vial prior to pouring the gel. As a rough
guide 100 ml of degassed 6% acrylamide gel will set in about
5 minutes at room temperature when gently mixed with 450
µl of freshly prepared 10% (w/v) ammonium persulphate and
200 µl TEMED. The setting time increases to about 10 minutes
if the TEMED volume is reduced to 100 µl and to approximately 15 minutes with 75 µl. The amount of catalysts may need
to be reduced under warm conditions.
Table below shows the volume of agarose solution required to
make the desired gel (1 mm thick) for each unit size. Adjustments are needed for 0.75, 1.5 or 2 mm spacers.
Model
Gel size (cm)
Volume (ml)
E4500
33x45
125
6. Care should be taken when selecting the pore size of the gel
to be used. The pore size or % of gel determines the resolving
ability given different sizes of protein. See table below which
details and which percentage of gel to use to separate the
sizes of proteins indicated.
Acrylamide percentage
Separating resolution
5%
60...220 kDa
7.5 %
30...120 kDa
10 %
20...75 kDa
12 %
17...65 kDa
15 %
15...45 kDa
17.5 %
12...30 kDa
For gel thicknesses thicker than 0.35 mm, first securely tape
the bottom of the gel with electrical tape then follow the instructions below.
1. Lay the plain glass plate on a flat surface and arrange the
spacers perfectly aligned with the edges of the plate.
2. Carefully place the notched glass plate on top of the plain
glass plate and clamp the plates together using bulldog clips
arranged along the edge of the glass plates, the pressure
clamps of these should be in line with the spacers.
3. Fill a syringe with the required gel mix. Be careful not to agitate or to introduce bubbles into the solution.
4. Position the syringe above one edge of the notch in a vertical
position. Steadily eject the gel solution along the notched
area moving the syringe spout smoothly from one side of the
notch to the other. The gel mix should form a continuous pool
along the top of the gel space move down between the glass
plates.
5. Be careful not to overfill the notched area. Fill gradually the
gel solution should be around half the height of the notches
on the notched glass plate. Also ensure not to under fill the
notched area as air bubbles are more likely to be introduced
between the glass plates. The boundary of the gel should migrate as a straight line. To prevent or expel bubbles, the glass
plates can be tapped lightly behind the moving gel boundary
to prevent any bubble formation.
6. When the gel boundary reaches the bottom of the glass
plates, remove all the surplus gel from the notched area with
the syringe. This will ensure that the gel mix doesn’t drip
from the bottom of the glass plates.
7. Insert the comb. If a square well comb is used, insert the
teeth making sure no bubbles are trapped. When using a
shark’s tooth comb, insert the flat face of the comb at a slight
angle to prevent bubbles from being trapped. A few drops of
gel mix can be added if necessary.
8. Carefully straighten the shark’s tooth comb so that it is parallel to the top of the gel plate and reaches 3 - 5mm below the
notched area.
9. For low percentage and DNA sequencing gels, leave to polymerise completely for at least 90 minutes. Low percentage
gels can be left to polymerise overnight. To prevent the ends
of the gel from drying out use wet tissues under a nesco film
seal.
Assembling the unit
1. Insert the lower buffer chamber into position.
2. Remove the bulldog clips and the bottom tape, if used, from
the glass plates.
3. Insert the glass plates behind the clamping bars and tighten
the screws. Do not over-tighten the gel plate clamping
screws as this may lead to the glass plate breakage and will
also make the insertion and removal of combs difficult.
4. Attach the leads to the buffer chambers.
5. Ensure that the buffer drainage tap is in the closed position.
6. Into the upper buffer chamber pour between 400 ml min. and
1000 ml max. of electrophoresis running buffer.
7. Into the bottom buffer chamber pour between 400 ml min.
and 1000 ml max. of electrophoresis running buffer. Important: do not fill over the maximum fill lines.
8. Prior to loading samples, flush out the wells with running
buffer to clear them of urea and debris.
Preparation of samples for loading
DNA Sequencing
1. The volume of sample depends on the capacity of the wells
(See Comb specifications page).
2. Heat the sequencing samples in a water bath or heating block
at 95°C for 3 minutes, place on ice and centrifuge at 12000
rpm for 3 minutes. Return to ice.
Denatured protein
The instructions given below are for denatured samples. For native samples, please consult a laboratory handbook.
1. Prepare the protein samples for loading. The volume of sample depends on the capacity of the wells.
2. Using a 0.5 ml micro-centrifuge tube or other convenient receptacle, combine the protein sample and 4 x sample buffer.
It is always advisable to use protein markers in one of the end
lanes to indicate sizes of bands. These should be prepared
according to the manufacturers instructions.
3. Heat the samples in a water bath or heating block for 2 minutes to denature the samples.
4. Centrifuge the samples in a micro-centrifuge for 20 seconds
at 12000 rpm. The protein samples are now ready to load.
Gel running and ending
Running the gel
1. Remove the square tooth comb before loading the samples. If
using a shark’s tooth comb, leave this in position as the comb
teeth will act as the wells.
2. Load the required volume of sample using a suitable loading
tip. If possible avoid taking the sample from the bottom of
the tube (particulate materials may cause streaking or smearing). Sample dispersion can be minimized by loading the sample directly onto the bottom of the well and keep it as a thin
layer.
3. Fit the safety lid ensuring it is positioned fully down over the
electrical connectors.
4. Connect and run the gel at the desired power setting. The
leads and electrical connectors are CE safe to 1500 V and users are advised not to exceed this voltage.
5. Typically for DNA sequencing gels, 45...55 W constant power
is advised. For other types of gel please consult a laboratory
handbook.
6. Turn the power supply off when the loading dye reaches the
bottom of the gel, sooner if your proteins are below 4 Kd in
size.
Ending the Run
1. Disconnect and turn off the power supply before removing
the leads.
2. Remove the safety lid by gripping the edges of the lid and
pushing down with your thumbs on the pegs located on the
top of the unit.
3. Separate the plates with a strong, thin, broad blade. Do not
force the glass plates apart at the notch as this may damage
the plates. The gel will usually stick to one of the plates and
can be removed by first soaking in buffer and then gently lifting with a spatula. For protein gels, the gel is now ready to be
stained with coomassie or silver stain or the proteins in the
gel can be transferred to a membrane by electroblotting for
specific band identification and further analysis.
24
E4xxx
Solutions for DNA sequencing
10 x DNA sequencing Buffer
• 164.0 g Tris-OH
• 27.5 g boric acid
• 7.45 g disodium EDTA
• add distilled water to a final volume of 1000 ml
Acrylamide stock
• 38% acrylamide
• 2% bis-acrylamide
8% Gel
• 40.4 g urea
• 27.0 ml water
• 16.8 ml 38/2 acrylamide
• 8.0 ml 10 x DNA sequencing buffer
6% Gel
• 40.4 g urea
• 31.2 ml water
• 12.6 ml 38/2 acrylamide
• 8.0 ml 10 x DNA sequencing buffer
5% Gel
• 40.4 g urea
• 33.5 ml water
• 10.5 ml 38/2 acrylamide
• 8.0 ml 10 x DNA sequencing buffer
If necessary mix by heating slightly, degassing is advised. Add 0.7 ml 10% ammonium persulphate, and 25 µl TEMED, and pour gel immediately.
E4xxx
25
Denaturing gradient system
Installation
1. Connect the heating element plug into the socket at the rear
of the control unit.
2. Connect the Pt100 temperature sensing probe plug at the rear
of the control unit.
3. Ensure the temperature is set to zero.
4. Connect the mains cable socket to the rear of the control
unit and connect to an external power source. Do not attempt to apply temperature to the tank without any buffer
present!
Operation
1. Fill the tank with buffer to the required level.
2. Ensure all the leads are connected in the correct positions as
described on page 4.
3. Switch on the control unit and ensure that the red power light
illuminates.
4. Set the required temperature on the control unit.
5. The heaters will remain on until the desired temperature has
been reached, at which point the unit will switch on and off
to maintain that temperature.
6. To obtain an accurate temperature of the buffer, it may be
advisable to attach a temperature strip panel to the front of
the tank.
Safety considerations
1. Should the sensor develop a fault or become disconnected the
heaters will automatically switch off so safeguarding against
gel over heating.
2. To replace the fuse isolate on the control unit from the mains
supply and open the fuse holder with a screwdriver blade.
The holder contains two fuses. Always use the recommended fuse and never replace it with a different one of different rating.
26
E4xxx
E4xxx
27
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Consort
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Tel: (+32) (0)14 41 12 79
Fax: (+32) (0)14 42 91 79
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