Labnet ENDURO GEL XL E0160 Manuel utilisateur

Lit M00078 Rev 3; September 2016

ENDURO™ GEL XL

User Manual

HORIZONTAL GEL ELECTROPHORESIS UNIT

E0160

E0160-230V

E0160-230V-UK

E160-CAN

About This Manual

This manual is designed to assist you in the optimal usage of your Enduro

GelXL. The manual is available in English, French, German, Italian, Portuguese,

and Spanish on our website at www.labnetinternational.com/document-center

TABLE OF CONTENTS

I. MAINTENANCE 1

II. OPTIONS AND SPECIFICATIONS 2

Components and Accessories 2

Specifications 3

III. OPERATING INSTRUCTIONS 3

Preparation of the Agarose Gel and Electrophoresis Buffer – DNA 3

Preparation of the Agarose Gel and Electrophoresis Buffer - RNA 5

Casting the Gel 6

Removing the Comb 7

Loading the Samples onto the Gel 8

Electrical Connections to the Safety Lid 8

Sample Electrophoresis 9

Detection and Documentation of Separated Fragments 10

IV APPENDICES 11

Buffers for Electrophoresis 11

Physical Properties of Electrophoretic Plastics 12

V REFERENCES 12

DECLARATION OF CONFORMITY 17

WARRANTY 18

I. MAINTENANCE

Please handle the unit with care:

Do not expose the unit or its accessories to temperatures above

60°C.

Do not expose the unit to organic solvents.

Do not clean the unit with abrasive cleaners or cleaning aids.

In most cases, rinsing with deionized water will sufficiently clean the unit.

For heavier dirt, use a mild cleansing solution such as dish soap (alkaline

cleansers are not recommended). Hand wash and dry with a soft cloth.

To remove residual ethidium bromide, occasionally soak the unit in 1%

commercial bleach solution for 16 hours. Rinse well.

PLEASE NOTE: The degradation of acrylic due to solvents may result in

substantial discoloration, cracking, warpage, or etching of the

electrophoresis unit.

Do not apply any of the following solvents: benzene, xylene,

toluene, chloroform, carbon tetrachloride, alcohols, phenols, ketones,

or esters.

Do not expose the ABS combs supplied with this unit to

formaldehyde for extended periods. When casting gels containing

formaldehyde, remove the combs promptly upon hardening of the gel

and rinse completely with deionized water.

Elimination of RNase Contamination

Should treatment of the unit to eliminate RNase contamination be desired, clean

the unit with a mild detergent as described above followed by soaking for 10

minutes in a solution of 3 % hydrogen peroxide, and then for 1 hour in 0.1 % DEPC

(diethyl pyrocarbonate). Pour out final rinse, and air dry.

CAUTION: DEPC is a suspected carcinogen; handle with care.

Alternatively, soak the unit and accessories in freshly made 2.2 mM acetic

anhydride treated water (200 µl/liter) for at least five minutes. Solutions for RNA

work (electrophoresis buffers, etc) may be made from the same acetic anhydride

treated water as well.

WARNINGS:

CAUTION! Injury, damage to equipment, or property, may result if used in a

manner not specified by the manufacturer.

CAUTION! A pinch hazard exists between the plastic enclosure and the shaking

head.

CAUTION! NOT for use with flammable liquids.

II. OPTIONS AND SPECIFICATIONS

A. Components and Accessories

Catalog # Description

E0160 ENDURO Gel XL Complete Electrophoresis System

Comes complete with 1) 12.5 x 12 cm, 2) 12.5 x 6 cm UV Transmittant casting

trays, casting stand with divider, and four 1.0 mm thick 28/14 reversible tooth

combs, power cord and manual.

Accessories

Catalog #

Description

E0161

(1)12.5 x 12cm UV Transmittant Casting Tray

E0162

(2) 12.5 x 6 cm UV Transmittant Casting Tray

E0163

(4) 6 x 6cm UV Transmittant Casting Tray

E0164

(2) 1 mm x 14/28 tooth Reversible Comb

E0165

(

2) 1mm 5/8 tooth Reversible comb

E0166

Micro casting set - (4) 6 x 6cm UV Transmittant Casting Tray, 2) 1mm

5/8 tooth Reversible combs, Casting stand with divider

E0167

Casting Stand with divider

E0168

Standard casting set – (1) 12.5 x 12cm tray, (2) 12.5x6cm trays, (4)

14/28 tooth multi-channel compatible combs, casting stand with

divider

R1000

100BP

Molecular Weight marker 100 bp

R1000

1KB

Molecular Weight marker 1 Kb

B. Specifications

Unit dimensions 24.5 x 17.0 x 6.2 cm

Gel dimensions 12.5 x 12.0 cm

Maximum sample capacity: 112 samples (4 combs, 26

samples each)

Buffer Capacity: 300 ml

Distance Between Electrodes: 13.5 cm

Electrophoresis Tank

Overall dimension 18.3 × 16.4 × 5.6 cm

Material characteristic UV transmitting (50% at 254nm,

80% at 312nm)

Solution volume 300ml (includes buffer and gels)

Safety Lid

Overall dimension 19.7 × 16.9 × 3.8 cm

Material characteristic UV non-transmitting Polycarbonate

Power Supply

Overall dimension 7.5 × 17.0 × 6.2 cm

Weight 410 g

Input Voltage AC100 - 240V, 50/60Hz

Output Voltage 10 to 150 volts; Constant peak

voltage of 150V

Output Amperage 10 to 400 mA

Maximum Wattage 45 W

Timer 99 hours 59 min, and continuous

model

Safety Switch Micro-sensor (hall) in the Power

Supply. No output without safety lid,

Memory Function Automatic memory (the last used V

and T)

III. OPERATING INSTRUCTIONS

A. Preparation of the Agarose Gel and Electrophoresis Buffer -

DNA

1. Select the percentage gel necessary to effectively resolve your

sample, using Table 1 as a guideline.

Table 1: Gel Concentrations and Resolving Ranges

Table taken from Sambrook, J., Fritsch, E.F., & Maniatis, T. (1989)

Molecular Cloning, A Laboratory Manual, 1, 6.8 613.

2. Weigh an appropriate quantity of agarose (0.3 % means 0.3 g of

agarose per 100 ml of gel volume) and place it into a 250 ml flask.

Note a 4mm gel will use 100 mls of agarose solution.

3. Make 500 ml of either 1X TAE or 1X TBE electrophoresis buffer

(see below).

Electrophoresis Buffers

The two most commonly used buffers for horizontal

electrophoresis of double stranded DNA in agarose gels are Tris-

Acetate-EDTA (TAE) and Tris-Borate-EDTA (TBE). While the

Concentration of Efficient Range of

Agarose in Gel Separation of Linear DNA

(% w/V) (Kb)

0.3% 5 - 60

0.6% 1 - 20

0.7% 0.8 - 10

0.9% 0.5 - 7

1.2% 0.4 - 6

1.5% 0.2 - 3

2.0% 0.1 - 2

resolving powers of these buffers are very similar, the relative

buffer capacities are very different, conferring different run

attributes which are summarized below:

TAE: Tris-acetate has traditionally been the more commonly

used buffer. However, its relatively low buffer capacity will

become exhausted during extended electrophoresis,

making buffer recirculation necessary in runs exceeding

140 mA-hours. Potential advantages of using TAE buffer

over TBE buffer include superior resolution of supercoiled

DNA and approximately 10 % faster migration of double-

stranded linear DNA fragments (1) .

TBE: Tris-borate’s significantly greater buffering capacity and its

relatively low current draw eliminates the need for

recirculation in all but the most extended runs (> 300 mA-

hours). TBE buffer systems are not recommended when

fragments are to be recovered from the gel after

electrophoresis.

4. Add ethidium bromide to the diluted electrophoresis buffer to a

final concentration of 0.5 µg/ml.

NOTE: The addition of ethidium bromide to both the gel and the

running buffer will result in maximum detection levels by

providing high levels of sample fluorescence with an evenly

low level of background.

5. Add 6.6 ml of the 1X electrophoresis buffer containing ethidium

made in step 4 per millimeter of gel thickness desired, up to a

maximum to 100 ml, to the flask containing the agarose. A 100

ml gel solution will make a 7.6 mm thick gel. Thinner gels may be

made, however care must be taken that the wells are deep

enough to accommodate the desired sample volume.

Catalog # Comb Description Well Width Sample Volume 1mm

E0167 1 mm, 14 tooth 5mm 5ul

E0167 1 mm, 28 tooth 2.5mm 2.5ul

E0168 1 mm, 5 tooth 8mm 8ul

E0168 1mm, 8 tooth 4mm 4ul

6. Make note of the total solution volume so that degree of

evaporation can be determined and corrected for.

7. Heat the agarose slurry in a microwave oven for 90 seconds.

Swirl the flask to make sure any grains sticking to the walls enter

into the solution. Undissolved agarose appears as small "lenses"

floating in the solution. Heat for an additional 30 - 60 seconds.

Re-examine the solution and repeat the heating process until the

agarose completely dissolves.

8. Add deionized water to replace any volume lost through

evaporation during the heating process.

Proceed to Section C, Step 1, “Casting the Gel” on page 12.

B. Preparation of the Agarose Gel and Electrophoresis Buffer - RNA

RNA molecules are separated by electrophoresis through denaturing gels prior to

analysis by northern hybridization. Agarose gels containing formaldehyde(1, 2, 3) are

commonly used for RNA electrophoresis. Presented below is a general protocol

for electrophoresis of RNA using formaldehyde gels.

CAUTION! All equipment and solutions used in the following protocol

should treated with DEPC (diethyl pyrocarbonate) or acetic

anhydride prior to use to inhibit RNase activity (see Section II,

page 4 for protocol). It is recommended that dedicated

solutions be made solely for RNA work to minimize the risk of

sample degradation due to RNase activity.

NOTE: Staining RNA samples with ethidium bromide has been

reported to reduce sample blotting efficiency. Therefore, if

samples are to be analyzed by northern hybridization after

electrophoresis, run a duplicate lane(s) for staining, or

minimize the exposure of RNA samples to ethidium bromide

by following the post-electrophoresis staining protocol on page

12.

The following protocol will make 50 ml of a 1.5 % agarose gel containing 1X MOPS

[3-(N-morpholino)-propanesulfonic acid]-Acetate-EDTA (MAE) buffer and 2.2 M

formaldehyde, resulting in a 7.5 mm thick gel:

1. Weigh out 0.5 g of agarose, and place into a 125 ml flask.

2. Add 43.5 ml of DEPC (or acetic anhydride) treated water.

3. Make note of the total solution volume so that degree of

evaporation can be determined and corrected for.

4. Heat the agarose slurry in a microwave oven for 60 seconds.

Swirl the flask to make sure any grains sticking to the walls enter

into the solution. Undissolved agarose appears as small "lenses"

floating in the solution. Heat for an additional 30 - 60 seconds.

Re-examine the solution and repeat the heating process until the

agarose completely dissolves.

5. Add deionized water to replace any volume lost through

evaporation during the heating process.

6. Allow the solution to cool to 60oC. Place the flask in a hood and

add 5 ml of 10X MAE buffer (see Appendix A for recipe), and 1.5

ml of 37 % formaldehyde.

CAUTION: Formaldehyde vapors are toxic. Gel

preparation should take place in a hood and solutions and gels

containing formaldehyde should be kept covered when

possible.

Proceed with Section C, Step 1, “Casting the Gel” on page 12.

C. Casting the Gel

1. Place the gel casting stand on a lab bench.

CAUTION! Cast agarose gels containing formaldehyde in a hood.

2. Insert the gel casting tray into the casting Stand. If you are using

the 12 x 6 cm gels place the spacer in the center of the casting

Stand, then insert the two 12 x 6 cm landscape gel trays (see

direction 2 below).

3. When the gel solution has cooled to approximately 55oC, slowly

pour it into the gel tray. If hotter gel

solutions are routinely poured

the tray may warp over time.

4. If bubbles form on the surface of the gel upon pouring, use the

comb to either pop them or lightly brush them to the sides of the

gel. If large bubbles are allowed to harden within the gel, they

may cause artifacts to occur during electrophoresis.

5. Insert one or more combs by placing them into the slots in the

casting stand. For best results, place the comb in the slot nearest

the end of the casting fixture. If two combs are desired, place the

second in the center comb slot.

6. Allow the gel to harden undisturbed for at least 30 minutes.

D. Removing the Comb

1. When the gel is solidified and fully opaque, carefully remove the

comb with a gentle wiggling, upward motion. If the comb is

difficult to remove or if a low percentage gel is being used, overlay

the comb area with a small volume of 1X electrophoresis buffer to

preserve the integrity of the wells. Check the wells to ensure their

bases are intact.

CAUTION: Prolonged exposure of the combs supplied to gels containing

formaldehyde will cause them to degrade. Be sure to remove

the comb(s) from formaldehyde gels as soon as gel hardening

is complete and rinse them well prior to storage.

If a gel is not to be used immediately after preparation, remove it from the

casting fixture and place it in a plastic bag or container and submerge in 1X

electrophoresis buffer containing 1 mM NaN3. Store at +4oC.

E. Loading the Samples onto the Gel

1. Remove the casting tray containing the hardened agarose gel

from the casting fixture by lifting the ends. Place the tray and gel

into the main unit assembly such that the samples wells are on

the same end as the negative (black) electrode.

2. Fill the unit with the remaining 1X electrophoresis buffer

containing ethidium bromide made previously (or 1X MAE buffer

for RNA gels), covering the gel to a depth of 1-5 mm.

Approximately 300 ml of buffer will be required.

NOTE: Use of the same batch of electrophoresis buffer for both the

gel and the running buffer is very important. Slight variations

in buffer composition between gel and running buffer may

result in ionic or pH gradients that can significantly impact the

mobility of the samples.

3. Pre-run RNA gels at 100 V for five minutes prior to loading the

Ce manuel convient aux modèles suivants

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