Consort E3100 User manual

Consort
MANUAL
E3100
E3200
E3300
E3400
E3500
E3600
July 2009
Distributor:
Topac Inc
231 CJC Highway
Cohasset
MA 02025
Tel [phone]
Fax [phone]
www.topac.com
email [email protected]om

Table of contents
Warranty
...............................................................
.
1
Safety precautions
.....................................................
2
General care and maintenance
......................................
3
Environmental conditions
............................................
3
Fitting electrode cables
..............................................
4
Fitting loading guides
.................................................
4
Gel preparation
........................................................
5
Gel pouring with casting dams
......................................
6
Gel pouring with model E3100
.......................................
6
Gel pouring using tape method
......................................
7
Gel pouring using the flexicaster
....................................
7
Running the gel
........................................................
8
Gel staining and viewing
.............................................
8
Solutions
...............................................................
.
9
Troubleshooting
........................................................
10

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1
Warranty
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 defec-
tive 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 out-
lined 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 incor-
rect 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 prod-
ucts without prior notice. This will enable us to implement devel-
opments 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.

2
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Safety precautions
Safety precautions
•
When used correctly, these units pose no health risk.
•
However, these units can deliver dangerous levels of electric-
ity and are to be operated
only
by quali ed personnel follow-
ing the guidelines laid out in this manual.
•
Anyone intending to use this equipment should
read
the com-
plete 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 suspect-
ed carcinogen. Wear effective protective clothing and follow
recommended handling and disposal procedures.
•
Polymerised gels contain some unpolymerised monomer. Han-
dle 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 pro-
duced depends on the composition of the buffer employed.
To disperse these gases make sure that the apparatus is run in
a well ventilated area.

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3
3
Environmental conditions
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 conden sation must be
expected”.
General care and maintenance
General care and maintenance
•
Units are best cleaned using warm water and a mild deter-
gent.
W
ater 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: w
arm
water with a mild concentration of soap or other mild de-
tergent (compatible detergents include dish washing liquid,
hexane and aliphatic hydrocarbons). The units should not be
left to in detergents for more than 30 minutes.
•
T
he units should never come into contact with the fol-
lowing 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 per-
oxide (H
2
O
2
) 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.
consult the instructions for use with acrylic gel tanks.

4
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Fitting electrode cables
Fitting electrode cables
1
.
Note the position of the lid on the unit. This shows the cor-
rect 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
no gap between the lid and the leading edge of
the cable fitting.
4
.
Refit the lid.
Fitting loading guides
Fitting loading guides
These can be fitted to enhance visibility of the wells if desired.
They can be fitted to the white vinyl platform sheet or to the
unit itself.
1
.
Seat the tray in the unit and note the position of the comb
grooves. The samples run black to red but the trays can be
used frontward or backwards so ensure that the comb grooves
closest to the black electrode are marked.
2
.
Remove the tray.
3
.
Peel the back off of the loading guide and carefully apply the
loading guide directly to the gel platform.

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5
5
Gel preparation
Gel preparation
1
.
Table below shows the volume of agarose solution required to
make the desired agarose gel (5 mm thick) for each unit tray
size. For a standard 0.7 % agarose gel, add 0.7 g of agarose to
100 ml of 1x TAE or TBE solution. The same 1x solution should
be used in the tank buffer solution.
Model
Gel size (cm)
Volume (ml)
E3100
10x8
40
E3200
7x7
25
7x10
35
E3300
10x7
35
10x10
50
E3400
15x7
53
15x10
75
15x15
113
E3500
20x10
100
20x20
200
E3600
26x16
208
26x24
312
26x32
416
2
.
Add the agarose powder to a conical flask.
3
.
Add the appropriate amount of 1 x TAE or TBE solution from
the table above. To prevent evaporation during the dissolving
steps below, the conical flask should be covered with para-
film.
4
.
Dissolve the agarose powder by heating the agarose either on
a magnetic hot plate with stirring bar or in a microwave oven.
If using the microwave method, the microwave should be set
at around a 400 W or medium setting and the flask swirled
every minute. The solution should be heated until all crystals
are dissolved. This is best viewed against a light background.
Crystals appear as translucent crystals. These will interfere
with sample migration if not completely dissolved.
5
.
The gel must be cooled to between 50°C and 60°C degrees
before pouring.

6
6
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Gel pouring
Gel pouring
with casting dams
with casting dams
Gel pouring with casting damsGel pouring
Gel pouring with casting damsGel pouring
1
.
Fit the casting dams over each end of the tray and place onto
a level surface. The dams should be fitted so that there is no
gap between the sides of the tray and the groove in the dams.
This will ensure that there is no possibility of gel leakage.
2
.
Place the comb(s) in the grooves. Each tray has more than
one comb grove so that multiple combs can be used. Using
multiple combs increases sample number available per gel
but decreases run length and care must be taken to ensure
that samples from the first wells do not migrate into the lanes
of the second comb wells.
3
.
Pour in the agarose carefully so as not to generate bubbles.
Any bubbles that do occur can be smoothed to the edge of the
gel and dispersed using a pipette tip.
4
.
Allow the agarose to set, ensuring that the gel remains undis-
turbed.
5
.
Carefully remove the gel casting gates and comb and transfer
the gel including tray to the main tank.
the gel including tray to the main tank.
Gel pouring
Gel pouring
with model E3100
with model E3100
Gel pouring with model E3100Gel pouring
Gel pouring with model E3100Gel pouring
1.
Fit the casting gates into the grooves in the unit which are 10
mm from the platinum wire. Ensure that these are seated as
deeply as possible. This will ensure that a good seal is formed
and that there is no possibility of gel leakage. A small amount
of vaseline or sealing grease on the bottom and side edges of
of vaseline or sealing grease on the bottom and side edges of
the gates will give further leakage protection.
2
.
Place the comb(s) in the grooves. Each tray has more than
one comb grove so that multiple combs can be used. Using
multiple combs increases sample number available per gel
but decreases run length and care must be taken to ensure
that samples from the first wells do not migrate into the lanes
of the second comb wells.
3
.
Pour in the agarose carefully so as not to generate bubbles.
Any bubbles that do occur can be smoothed to the edge of the
gel and dispersed using a pipette tip.
4
.
Allow the agarose to set, ensuring that the gel remains undis-
turbed.
5
.
Carefully remove the gel casting gates and comb and transfer
the gel including tray to the main tank.

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7
Gel pouring
Gel pouring
using tape method
using tape method
1
.
Autoclave or plastic backed general tape should be used. A
length 5 cm longer than the width of each end of the tray
should be cut. One length should be placed over one end of
the tray folded and the edges sealed securely. Repeat for the
other end and place onto a level surface for gel pouring.
2
.
Place the comb(s) in the grooves. Each tray has more than
one comb grove so that multiple combs can be used. Using
multiple combs increases sample number available per gel
but decreases run length and care must be taken to ensure
that samples from the first wells do not migrate into the lanes
of the second comb wells.
3
.
Pour in the agarose carefully so as not to generate bubbles.
Any bubbles that do occur can be smoothed to the edge of the
gel and dispersed using a pipette tip.
4
.
Allow the agarose to set, ensuring that the gel remains undis-
turbed.
5
.
Carefully remove the gel casting gates and comb and transfer
the gel including tray to the main tank.
the gel including tray to the main tank.
Gel pouring using the flexicaster
Gel pouring using the flexicaster
1
.
Level the  exicaster base by adjusting the feet so that the
bubble is exactly central.
2
.
Insert the desired length tray into the  exicaster such that
one end of the tray is pushed up and seals against the silicone
mat of the permanent end of the  exicaster.
3
.
Position the movable end of the  exicaster so that the sili-
cone mat is pushed against the other end of the tray.
4
.
Turn the cam so that the silicone mat tightly seals against
the side of the tray. Pour in the agarose carefully so as not to
generate bubbles. Any bubbles that do occur can be smoothed
to the edge of the gel and dispersed using a pipette tip.
5
.
Allow the agarose to set, ensuring that the gel remains undis-
turbed.
6
.
Carefully remove the gel casting gates and comb and transfer
the gel including tray to the main tank.

8
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Gel staining and viewing
Gel staining and viewing
All trays and the E3100 unit allow staining to be performed with-
out removing the gel from the tray if this is preferred.
1
.
Transfer the gel to a vessel containing the appropriate vol-
ume of 0.5 µg/ml ethidium bromide stain for 15...30 minutes,
see solutions for stock stain concentration and adjust to the
volume used accordingly. The entire gel should be covered.
(Caution: ethidium bromide is a suspected carcinogen and the
necessary safety precautions should be undertaken).
2
.
De-stain the gel for 10–30 minutes in distilled water again
ensuring the gel is completely immersed.
3
.
Rinse the gel twice for a couple of seconds with distilled wa-
ter.
4
.
Transfer the gel to a UV Transilluminator.
5
.
The samples will often appear as brighter, clearer bands when
photographed or viewed using a gel documentation system.
However if the gel bands are too faint then the staining pro-
cedure should be adjusted so that there is less de-staining.
If there is too much background then the staining procedure
should be adjusted so that there is more de-staining.
Running the gel
Running the gel
1
.
Mix the sample to be loaded with sample buffer (see solutions
for common sample buffers). Usually 3 µl of sample buffer is
adequate but less may be used with sample volumes of less
than 10 µl.
2
.
Fill the unit with buffer until the gel is just flooded with buff-
er. This will give the fastest resolution times. For enhanced
quality of resolution of sample, fill the unit to 5 mm above
the gel.
3
.
Load the samples into the wells using pipettes. Multi-channel
pipettes can be used for loading samples with MultiPipette
compatible combs, see listing in accessories for identification
of these.
4
.
Carefully place the lid on the tank and connect to a power
supply.
5
.
Typically gels are run at between 90 and 150 V. However,
maximum voltage is indicated on the serial badge of each
unit. It should be noted that a higher voltage generally give
faster but poorer quality sample resolution.

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9
9
Solutions
Solutions
1x TAE 40 mM tris (pH 7.6), 20 mM acetic acid, 1 mM EDTA
•
50 x (1l) dissolve in 750 ml distilled water:
•
242 g tris base (FW = 121)
•
57.1 ml glacial acetic acid
•
100 ml 0.5 M EDTA (pH 8.0).
•
Fill to 1 litre with distilled water.
1x TBE 89 mM tris (pH 7.6), 89 mM boric acid, 2 mM EDTA
•
10 x (1l) dissolve in 750 ml distilled water:
•
108 g tris base (FW = 121)
•
55 g boric acid (FW = 61.8)
•
40 ml 0.5 M EDTA (pH 8.0)
•
Fill to 1 litre with distilled water.
Sample Loading Dye
•
10x sample buffer stock consists of 50% glycerol, 0.25%
bromophenol blue, and 0.25% xylene cyanole FF in 1x TAE
buffer. Only 1–10 ml of the 10x loading dye should be pre-
pared.
Ethidium Bromide Solution
•
Add 10 mg of Ethidium Bromide
to 1 ml distilled water.

10
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Troubleshooting
Troubleshooting
Equipment problem
•
Bubbles do not appear on the electrodes:
Ensure that the power supply and the whole electric assembly
is operating properly.
•
Melted agarose leaks when casting:
Ensure the agarose is not too hot when poured.
Ensure that the sealing surfaces of the running tray and the
gel casting gates are clean.
Ensure that the ends of the running tray are  at and free of
Ensure that the ends of the running tray are  at and free of
nicks.
Electrophoresis problem
•
Sample well deformed:
Allow the gel to set for a minimum of 30 minutes.
Leave comb in position until gel returns to room temperature
before removing.
Remove the comb both slowly and at a slight angle to prevent
gel from breaking.
Avoid damaging the well with the pipette when loading the
sample. Aim for the centre of the well and avoid damaging
the bottom of the well with the pipette tip.
•
Samples leak underneath the gel upon loading:
The bottom of the wells were torn when the comb was re-
moved. To avoid this tearing, carefully wiggle the comb to
free the teeth from the gel.
•
Distorted sample wells:
Incomplete polymerisation produces poorly defined wells.
De-gas gel solution prior to casting and increase ammonium
persulphate and TEMED concentrations.
•
Samples do not run straight:
Comb may be warped and should be replaced.
Running tray may be warped and should be replaced. Reduce
the voltage.
Choose a buffer with suitable ionic strength and buffering
capacity.
•
“Smiling” along one edge of the gel:
Gel was not level when cast or run. Use a gel levelling table
to ensure that the apparatus is level prior to gel casting and
electrophoresis.

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11
•
Bromophenol blue dye turns yellow:
Check pH of buffer during electrophoresis. (pH change).
Ensure Tris base and not Tris-HCl was used.
Mix the buffer periodically during electrophoresis. Connect a
pump to circulate the buffer.
•
Double-banded pattern
Ensure the comb is vertical during casting so that the well
shape is not distorted.
Decrease the buffer level to 1 mm above the top of the gel.
This will reduce the temperature gradient through the gel.
Increase concentration of the sample and use a thin (2 to 3
mm) gel with a thin (1 mm) comb.
•
"Tailed" bands (excessive  uorescence appearing above the
band)
Reduce DNA in the sample.
Reduce the protein and/or glycerol in the sample.
•
Poor band resolution
Add  coll, glycerol, or sucrose to the sample loading buffer
to ensure that the sample layers on the bottom of the well.
Ensure sample is completely dissolved.
Reduce voltage, sample concentration, or sample volume.
Ensure there is at least 1 mm of gel below the bottom of the
comb to prevent samples from leaking out the bottom of the
well.
Reduce salt concentration of the sample. High salt concentra-
tions can cause "pinched" lanes, smeared lanes, arched dye
front and slow migration.
Check enzyme activity as it may require longer digestion or
different restriction buffer.
Prepare fresh sample if nuclease contamination is suspected.
Choose agarose with low endosmosis value.
•
Gel melts or softens near sample wells.
Caused by a combination of pH drift and high temperature.
Circulate or remix buffer periodically or reduce the voltage.

12
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DECLARATION OF CONFORMITY
We declare under our sole responsibility that the product
Horizontal Apparatus
content of the type numbers
E3100, E3200, E3300, E3400, E3500, E3600
to which this declaration relates is in conformity
with the following standards
EN61010
LOW VOLTAGE DIRECTIVE 73/23/EEG
EN50081-1
EN50082-1
EN60555-2
EMC DIRECTIVE 89/336/EEG
Turnhout, June 25, 2009
CONSORT nv
Tel (++32)(14)41 12 79
Parklaan 36
Fax (++32)(14)42 91 79
B-2300 Turnhout
E-mail: [email protected]