Consort E3100 Bedienungsanleitung

Consort

MANUAL

E3100

E3200

E3300

E3400

E3500

E3600

July 2009

Distributor:

Topac Inc

231 CJC Highway

Cohasset

MA 02025

Tel 781 740 8778

Fax 781 740 8779

www.topac.com

email [email protected]om

Table of contents

Warranty

...............................................................

Safety precautions

.....................................................

General care and maintenance

......................................

Environmental conditions

............................................

Fitting electrode cables

..............................................

Fitting loading guides

.................................................

Gel preparation

........................................................

Gel pouring with casting dams

......................................

Gel pouring with model E3100

.......................................

Gel pouring using tape method

......................................

Gel pouring using the flexicaster

....................................

Running the gel

........................................................

Gel staining and viewing

.............................................

Solutions

...............................................................

Troubleshooting

........................................................

E3xxx

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.

E3xxx

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

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.

E3xxx

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

•

Units are best cleaned using warm water and a mild deter-

gent.

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.

•

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

) 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.

E3xxx

Fitting electrode cables

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.

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.

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.

Refit the lid.

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.

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.

Remove the tray.

Peel the back off of the loading guide and carefully apply the

loading guide directly to the gel platform.

E3xxx

Gel preparation

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

E3200

7x7

7x10

E3300

10x7

10x10

E3400

15x7

15x10

15x15

113

E3500

20x10

100

20x20

200

E3600

26x16

208

26x24

312

26x32

416

Add the agarose powder to a conical flask.

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.

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.

The gel must be cooled to between 50°C and 60°C degrees

before pouring.

E3xxx

Gel pouring

with casting dams

Gel pouring with casting damsGel pouring

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.

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.

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.

Allow the agarose to set, ensuring that the gel remains undis-

turbed.

Carefully remove the gel casting gates and comb and transfer

the gel including tray to the main tank.

Gel pouring

with model E3100

Gel pouring with model E3100Gel pouring

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

the gates will give further leakage protection.

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.

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.

Allow the agarose to set, ensuring that the gel remains undis-

turbed.

Carefully remove the gel casting gates and comb and transfer

the gel including tray to the main tank.

E3xxx

Gel pouring

using tape method

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.

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.

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.

Allow the agarose to set, ensuring that the gel remains undis-

turbed.

Carefully remove the gel casting gates and comb and transfer

the gel including tray to the main tank.

Gel pouring using the flexicaster

Level the  exicaster base by adjusting the feet so that the

bubble is exactly central.

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.

Position the movable end of the  exicaster so that the sili-

cone mat is pushed against the other end of the tray.

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.

Allow the agarose to set, ensuring that the gel remains undis-

turbed.

Carefully remove the gel casting gates and comb and transfer

the gel including tray to the main tank.

E3xxx

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.

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).

De-stain the gel for 10–30 minutes in distilled water again

ensuring the gel is completely immersed.

Rinse the gel twice for a couple of seconds with distilled wa-

ter.

Transfer the gel to a UV Transilluminator.

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

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.

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.

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.

Carefully place the lid on the tank and connect to a power

supply.

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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