LFVERTICAL

Instruction Manual

Large Format
Vertical
Electrophoresis Systems

Catalogue Numbers

CSQ20
CSQ33

V03.03.15

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Contents:-
Page
1) Safety Instructions 3
2) Packing Lists 4
3) Care and Maintenance 5
4) System details 6
5) Plate Preparation 7
6) Gel Preparation 8
7) Gel Selection 9
8) Gel Pouring 10
9) Assembling the unit 11
10) Run Conditions 12
11) Sample Preparation 13
12) Gel Running 14
13) References 15
14) Solutions 16
15) DNA Sequencing Solutions 18
16) Combs 19
17) Warranty 20

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SAFETY PRECAUTION

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 INSTRUCTION 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.
ACRYLAMIDE IS A POWERFUL NEUROTOXIN IN SOLUTION FORM.
POLYMERIZED GELS CAN CONTAIN SOME UNPOLYMERIZED SOLUTION AND
PROTECTIVE GLOVES AND CLOTHING MUST BE WORN.
THESE UNITS COMPLY WITH THE STATUTORY CE SAFETY DIRECTIVES:
73/23/EEC: LOW VOLTAGE DIRECTIVE: IEC 1010-1:1990 plus AMENDMENT
1:1992
EN 61010-1:1993/BS EN 61010-1:1993

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PACKING LISTS

CSQ20, CSQ33
Units include tank, lid, internal module and electrodes and include the following
accessories:-

Glass Plates Comb Spacers Cables

CSQ20 NOTCHED, 1 CSQ20-0.35-48 CSQ20-S0.35 CSL-CAB2
PLAIN, 1

CSQ33 NOTCHED, 1 CSQ33-0.35-48 CSQ33-S0.35 CSL-CAB2

PLAIN, 1

The packing lists should be referred to as soon as the units are

received to ensure that all components have been included. The unit
should be checked for damage when received. Please contact your
supplier if there are any problems or missing items.

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Care and Maintenance
Cleaning Large Format Vertical Units
Units are best cleaned using warm water and a mild detergent. Water at
temperatures above 60 0 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 dishwashing 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.
RNase Decontamination
This can be performed using the following protocol:-
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.

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Usage Guidance and restrictions

• Maximum altitude 2,000m.

• Temperature range between 4°C and 65°C.
• Maximum relative humidity 80% for temperatures up to 31OC decreasing linearly
to 50%
relative humidity at 40OC.
• Not for outdoor Use.

This 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”.

Setting up the Large Format Gel Tanks

Instructions for 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.
The unit is now ready to be used.

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Cleaning and Preparation of Glass Plates

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.
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.
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.
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.
The plain glass plate should be siliconised along the outer 1cm 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 as described above.
The horizontal gel pouring method described in this email 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.
The above procedures are not necessary every time a gel is poured.
After use, first, clean using a neutral detergent and a small brush and wahs with
distilled water, ethanol and acetone as described above.
DO NOT ALLOW ORGANIC SOLVENTS INCLUDING ACETONE AND
ALCOHOLS TO COME INTO CONTACT WITH THE PLASTIC
COMPONENTS OF THE MAIN UNIT.

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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 16 to 17 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 100ml of degassed 6% acrylamide gel will set in about 5 minutes
at room temperature when gently mixed with 450 l of freshly prepared 10% (w/v)
Ammonium persulphate and 200 l TEMED. The setting time increases to about 10
minutes if the TEMED volume is reduced to 100 l and to approximately 15 minutes
with 75 l.

The amount of catalysts may need to be reduced under warm conditions.

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Gel Volumes:

Table 1 below shows the total volume of gel solution required.

Table 1.

CSQ20 CSQ33
Total Gel volume Total Gel volume
for a 1mm thick for a 1mm thick
gel. gel.
For different thicknesses of gel, multiple the below amounts by the spacer
thickness.
Single – one gel 80ml Single – one gel 125ml

Gel Selection:-
For protein gels, 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 2 below which details which percentage of gel to use to separate the sizes
of proteins indicated.
Table 2.
Acrylamide Percentage Separating Resolution

5% 60 - 220 KD

7.5 % 30 - 120 KD

10 % 20 - 75 KD

12% 17 – 65 KD

15 % 15 -45 KD

17.5% 12 – 30 KD

Gel Pouring
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For Gel Thicknesses 0.25 and 0.35mm, follow the instructions below.
For Gel Thicknesses thicker than 0.35mm, 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, see Table 1. for gel volume required and
pages 16 to 18 for gel solutions. 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.

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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

Insert the lower buffer chamber into position.

Remove the bulldog clips and the bottom tape, if used, from the glass plates.
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.
Attach the electrical connectors to the buffer chambers.

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Run Conditions and Buffer Volumes
Ensure that the buffer drainage tap is in the closed position.

CSQ20
Into the upper buffer chamber pour between 250ml min and 500ml max of
electrophoresis running buffer.
Into the bottom buffer chamber pour between 250ml min and 500ml max of
electrophoresis running buffer.
IMPORTANT do not fill over the Maximum fill lines.

CSQ33
Into the upper buffer chamber pour between 400ml min and 1000ml max of
electrophoresis running buffer.
Into the bottom buffer chamber pour between 400ml min and 1000ml max of
electrophoresis running buffer.
IMPORTANT do not fill over the Maximum fill lines.

Prior to loading samples, flush out the wells with running buffer to clear them of
urea and debris.

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Preparation of DNA Sequencing samples for loading:
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 950C for 3
minutes, place on ice and centrifuge - 12,000xg for 3 minutes. Return to ice.

Preparation of denatured 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 (See Comb specifications page).

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 12,000 rpm. The

protein samples are now ready to load.

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Gel Running:
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; consult the comb
specifications page 19 for details on maximum sample volume for the comb and gel
thickness used. 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 1,500 Volts and users are advised not to exceed this
voltage.
5. Typically for DNA Sequencing gels, 45 -55 Watts 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 4Kd in size.

Ending the Run

Disconnect and turn off the power supply before removing the connectors.
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.
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.

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For DNA Sequencing gels, the gel should be attached to filter paper and dried. Please
consult a laboratory handbook for the further handling of Sequencing gels.

Disconnect the leads to the power supply and remove the lid. Then disconnect the
lead to the lower buffer chamber.

Remove the bottom tank and empty then position the bottom tank around the back of
the unit underneath the drainage tap. Open the valve and the buffer will then flow
into the bottom buffer tank.

Again empty the lower buffer tank and carefully discard the buffer, this cannot be re-
used.

References:-

1. Sambrook, Fritsch, and Maniatis, Molecular Cloning A Laboratory Manual,

Second Edition,
Cold Spring Harbor Laboratory Press, 1989.

2. Current Protocols in Molecular Biology, Greene Publishing Associates and

Wiley-Interscience,1989.

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Stock Solutions for SDS PAGE gels:-

Stock 30% Acrylamide Gel Solution:-

30.0 g acrylamide

0.8 g methylene bisacrylamide

Distilled Water to 100ml

.
Stock 4 X Resolving Gel Tris (1.5 M Tris HCl pH8.8, 0.4 % SDS)

To 110ml Distilled Water add 36.4 g of Tris base

Add 8ml of 10 % SDS

Adjust pH to 8.8 with 1N HCl

Adjust the final volume to 200ml with Distilled Water.

.
Stock 4 X Stacking Tris (0.5 M Tris HCL pH6.8, 0.4 % SDS)

To 110ml Distilled Water add 12.12 g of Tris base

Add 8ml of 10 % SDS

Adjust pH to 6.8 with 1N HCl

Stock 4 X Tris-glycine tank buffer - SDS

36 g Tris base

172.8 g glycine

Distilled Water to 3 L

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1 x Tris-glycine tank buffer - SDS

750ml of 4 X Tris-glycine reservoir buffer - SDS

30ml of 10 % SDS

Distilled Water to 3L

Add Distilled Water to a final volume of 200ml

10 % AP (ammonium persulphate solution)

0.1 g ammonium persulphate

1ml Distilled Water

TEMED

Stock 4 X Sample Buffer

4ml glycerol

2ml 2-mercaptoethanol

1.2 g SDS

5ml 4 X Upper Tris

0.03 g Bromophenol blue

Aliquot into 1.5ml microcentrifuge tubes. Store at -20°C.

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Stock Solutions for DNA Sequencing gels:-
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 1L

Acrylamide stock

38% Acrylamide
2% bis-acrylamide

8% gel CSQ33 Gel

40.4 g urea
27.0 ml water
16.8 ml 38/2 acrylamide
8.0 ml 10 x DNA Sequencing Buffer
6% CSQ33 Gel
40.4 g urea
31.2 ml water
12.6 ml 38/2 acrylamide
8.0 ml 10 x DNA Sequencing Buffer
5% CSQ33 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 25ul TEMED, and pour gel immediately.

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Combs:–
CSQ20:-

Sample
Volum e µl for
a 5m m thick
Code Description gel
CSQ20-0.25-24 Comb 24 sample, 0.25mm thick, Sharks tooth 7
CSQ20-0.25-48 Comb 48 sample, 0.25mm thick, Sharks tooth 3
CSQ20-0.35-24 Comb 24 sample, 0.35mm thick, Sharks tooth 9
CSQ20-0.35-48 Comb 48 sample, 0.35mm thick, Sharks tooth 5
CSQ20-1-24 Comb 24 sample, 1mm thick, Square tooth 40
CSQ20-1-48 Comb 48 sample, 1mm thick, Square tooth 20
CSQ20-1.5-24 Comb 24 sample, 1.5mm thick, Square tooth 60
CSQ20-1.5-48 Comb 48 sample, 1.5mm thick, Square tooth 30

CSQ33:-

Sample
Volum e µl for
a 5m m thick
Code Description gel
CSQ33-0.25-48 Comb 48 sample, 0.25mm thick, Sharks tooth 7
CSQ33-0.25-96 Comb 96 sample, 0.25mm thick, Sharks tooth 3
CSQ33-0.35-48 Comb 48 sample, 0.35mm thick, Sharks tooth 9
CSQ33-0.35-96 Comb 96 sample, 0.35mm thick, Sharks tooth 5
CSQ33-1-48 Comb 48 sample, 1mm thick, Square tooth 35
CSQ33-1-80 Comb 80 sample, 1mm thick, Square tooth 20
CSQ33-1.5-48 Comb 48 sample, 1.5mm thick, Square tooth 50
CSQ33-1.5-80 Comb 80 sample, 1.5mm thick, Square tooth 30

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Warranty

The Large Format Vertical Electrophoresis units have a warranty against

manufacturing and material faults of twelve months from date of customer receipt.

If any defects occur during this warranty period, your supplier will repair or replace
the defective parts free of charge.

This warranty does not cover defects occurring by accident or misuse or defects
caused by improper operation.

Units where repair or modification has been performed by anyone other than your
supplier or an appointed distributor or representative are no longer under warranty
from the time the unit was modified.

Units which have accessories or repaired parts not supplied by your supplier or it’s
associated distributors have invalidated warranty.

Your supplier cannot repair or replace free of charge units where improper solutions
or chemicals have been used. For a list of these please see the Care and
Maintenance subsection.

If a problem does occur then please contact your supplier.

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