Labnet ENDURO96 E1010-9611 Manual de usuario

Lit M00203 May 2012

ENDURO96

HORIZONTAL GEL BOXES

User Manual

E1010-9611

E1010-9611.5

E1010-9621

E1010-9621.5

Lit M00203 May 2012

Contents:-

Page

1) Safety Instructions 1

2) Packing Lists 2

3) Usage Guidance and restrictions 3

4) Care and Maintenance 3

5) Setting Up 4

6) Gel Preparation 5

7) Gel Pouring 6

8) Gel Running 7

9) Gel Staining 8

10) Solutions 9

11) Combs 9

12) Symbols & Conventions 10

13) Equipment Disposal 10

14) Declaration of Conformity 11

15) Warranty 13

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.

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

PACKING LISTS:

E1010-9611, E1010-9611.5, E1010-9621, E1010-9621.5

Units include tank, lid and electrodes and include the following accessories:-

Cat no.

Tray

Dams

Combs and

comb block

Cables

E1010-9611

E1010-96T 10 x

12cm (W x L)

E1010-CD

Pack of 2

E1010-9611-CB

E1107-EP

E1010-9611.5

E1010-96T 10 x

12cm (W x L)

E1010-CD

Pack of 2

E1010-9611.5-CB

E1107-EP

E1010-9621

E1010-96T 10 x

12cm (W x L)

E1010-CD

Pack of 2

E1010-9621-CB

E1107-EP

E1010-9621.5

E1010-96T 10 x

12cm (W x L)

E1010-CD

Pack of 2

E1010-9621.5-CB

E1107-EP

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.

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”.

Care and Maintenance:-

Cleaning Horizontal Units

Units are best cleaned using warm water and a mild detergent. Water at

temperatures above 600 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.

Setting up the Horizontal 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.

Instructions for 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.

The unit is now ready to be used.

Gel Preparation:-

1. Table 1 below shows the volume of agarose solution required to make the

desired agarose gel for each unit tray size. For a standard 0.7% agarose gel,

add 0.7grammes of agarose to 100 ml of 1x TAE or TBE solution. The same

1 x solution should be used in the tank buffer solution.

E1010-9611 & E1010-9611.5

Gel volume for a 5mm thick gel

60ml

2. Add the agarose powder to a conical flask.

3. Add the appropriate amount of 1x TAE or TBE solution from the table

above. To prevent evaporation during the dissolving steps below, the conical

flask should be covered with parafilm.

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 watt 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.

Gel Pouring:-

The range of units allows three different methods of gel casting:-

Casting Dams

Flexicaster

Traditional Tape

Using trays with Casting Dams:

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 undisturbed.

5. Carefully remove the gel casting gates and comb and transfer the gel

including tray to the main tank.

Using Traditional tape method:-

1. Autoclave or plastic backed general tape should be used. A length

5cm longer than the width of each end of the tray should be cut. One

length should be placed over one end of the tray and stuck 1cm in

from the tray edge. This should then be 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 undisturbed.

5. Carefully remove the gel casting gates and comb and transfer the gel

including tray to the main tank.

Running the Gel:-

1. Mix the sample to be loaded with sample buffer –see solutions for

common sample buffers. Usually 3ul of sample buffer is adequate but less

may be used with sample volumes of less than 10ul.

2. Fill the unit with enough buffer so that it will just cover the gels when

they are immersed. This will give the fastest resolution times. For enhanced

quality of resolution of sample, fill the unit to 5mm above the gel.

3. On the bench surface, load a small amount of running buffer to flood the

wells. Load the samples into the wells using a pipette. Multi-channel

pipettes can be used forloading samples into wells formed by MC compatible

combs, see listing in accessories for identification of these.

4. Once loaded, gently immerse the gels within the buffer, stacking them

carefully on top of each other. Replace lid and connect unit to the power

supply using cables.

5. Typically gels are run at between 90 and 150 volts. However, maximum

voltages are indicated on the serial badge of each unit. It should be noted

that higher voltages generally give faster but poorer quality sample

resolution.

Gel Staining and Viewing:-

The Multi Sub trays and the Mini Fast unit allow staining to be performed

without removing the gel from the tray if this is preferred.

1. Transfer the gel to a vessel containing the appropriate volume 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.

NOTE:- 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 water.

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 procedure 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.

References

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

Manual, Second Edition,

Cold Spring Harbor Laboratory Press, 1989.

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