Apogee 21054010 Bedienungsanleitung

Apogee Electrophoresis | 101 Kane Street |Baltimore, MD 21224 USA |apogeephoresis.com

INSTRUCTION MANUAL

Blot Transfer Systems

11 x 14 cm Format, #21054010

Suitable for the Horizon® 11.14 and H5

20 x 25 cm Format, #11056017

Suitable for the Horizon® 20.25 and H4

Apogee Electrophoresis | 101 Kane Street |Baltimore, MD 21224 USA |apogeephoresis.com

TABLE OF CONTENTS

Before You Begin 1.0

Important Information 1.1

Components 1.2

Instructions for Use 2.0

Gel Preparation After Electrophoresis 2.1

Applying Wicking Sheets 2.2

Applying the Membrane and Wicking Sheets 2.3

Capillary Transfer 2.4

References 3.0

Related Products 4.0

Care and Handling 5.0

Materials and Care 5.1

General Specifications 5.2

Technical Support and Service 5.3

Instructions for Return Shipment 5.4

Cleaning and Decontamination for Return Shipment 5.4.1

Notice Regarding the Return of Apparatus Products 5.4.2

Warranty 6.0

Decontamination Declaration 7.0

Horizon ®is a registered trademark of Apogee Designs, Ltd.

Apogee Electrophoresis | 101 Kane Street |Baltimore, MD 21224 USA |apogeephoresis.com

1.0 BEFORE YOU BEGIN

1.1 IMPORTANT INFORMATION

The Horizon®11.14 and 20.25 Blot Transfer Apparatus are designed for capillary transfer of nucleic

acids from agarose gels to membranes in Southern and Northern blots. Used in conjunction with the

appropriate blotting materials and membranes, the included trays and supports offer a simple and

efficient method for performing high quality blot transfers.

Blot transfer apparatus 11.14 is compatible with 11 x 14 cm horizontal gel electrophoresis units

including the Horizon 11.14 and the Model H5 apparatus.

Blot transfer apparatus 20.25 is compatible with 20 x 25 cm horizontal gel electrophoresis units

including the Horizon 20.25 and the Model H4 apparatus.

This manual provides instructions on the use of both blot transfer apparatus noted above.

For operating procedures for the Horizon 11.14, 20.25, H4, and H5 Apparatus, as well as general

guidelines, practical applications, and suggestions for agarose gel electrophoresis, please refer to the

instruction manuals. These systems have not been qualified for use in any human or animal diagnostic

or therapeutic application. Use for other than the intended use may be a violation of applicable law.

Please carefully follow the manual’s instructions. Do not alter equipment or operate with broken

components. Failure to adhere to these directions could result in personal and/or laboratory hazards

as well as invalidate the equipment warranty.

1.2COMPONENTS

The 11.14 and 20.25 blot transfer apparatus are designed for durability and ease of use. Each complete

apparatus includes the following items:

One molded buffer tray

One machined and bent acrylic tray insert

One machined and bent acrylic gel restrainer

One instruction manual

Gel Restrainer

Buffer Tray

Tray Insert

Figure 1: Components

Apogee Electrophoresis | 101 Kane Street |Baltimore, MD 21224 USA |apogeephoresis.com

2.0 INSTRUCTIONS FOR USE

2.1 GEL PREPARATION AFTER ELECTROPHORESIS

Refer to Figure 1 to identify the features and components in these instructions.

The capillary blot technique originally developed by Southern (1) has been modified and extended in a

number of ways since its original publication. Protocols with buffer compositions for DNA and RNA blots

can be found in a number of journal articles and laboratory manuals (2 thru 5).

All protocols require one or more soaking steps before actual blotting begins. During these steps, the

agarose gels are fragile and easily damaged; whenever possible, handle the gel using the UVT tray on

which it was cast.

To help prevent the gel from slipping off the UVT tray during handling, place the gel restrainer over the

UVT tray. Orient the gel restrainer so that the bent tabs block the open ends of the UVT tray (Figure 2).

All soaking steps can be carried out in the buffer tray. Transfer the gel, in the UVT tray, into the buffer

tray and cover the gel completely with the appropriate soaking solution. Rock the buffer tray and gel

gently and regularly during soaking steps. Remove the gel and thoroughly rinse the buffer tray with

deionized water before adding each new solution.

2.2 APPLYING WICKING SHEETS

1. When all soaking steps are complete, remove the gel, in the UVT tray or gel plate, to a clean

work surface. Use the gel restrainer as needed while handling the gel in the UVT tray.

2. Wet a wicking sheet in the solution to be used for capillary transfer.

3. Place the wicking sheet on the gel. If using a UVT tray, align the edges of the wicking sheet

against the side rails of the tray with the overlap of the wicking sheet equal at the top and

bottom of the gel. The wicking sheet should run 90 degrees to the side rails of the UVT tray so

that the overlap does not run up the side rails.

Note: Do not trap air pockets between the gel and wicking sheet. Remove trapped air by lifting and

replacing the wicking sheet or by gently finger. Small bubbles in the wells will not affect transfer.

Figure 2: Gel restrainer in position over UVT tray

Apogee Electrophoresis | 101 Kane Street |Baltimore, MD 21224 USA |apogeephoresis.com

4. After the wicking sheet is in position, place the tray insert into a UVT tray at 90 degrees to each

other so that the gel is centered between, but not touching, the rounded edges of the insert

tray. Again, be careful not to trap air pockets between the tray insert and the wicking sheet

(Figure 3). With a gel plate just center the gel on the wicking sheet and insert the tray.

5. Hold the UVT tray (or gel plate) and tray/insert assembly securely but gently together and invert

the entire assembly.

6. Carefully remove the UVT tray or gel plate, leaving the gel in place on the wicking sheet on the

now inverted insert tray (Figure 4).

Note: The wicking sheet runs over the insert tray legs.

Figure 3: Gel and wicking sheet on tray insert

Figure 4: Inverted insert tray

Apogee Electrophoresis | 101 Kane Street |Baltimore, MD 21224 USA |apogeephoresis.com

2.3 APPLYING THE MEMBRANE AND BLOTTING PADS

Nitrocellulose and PVDF (positively-charged nylon) membranes are commonly available and both work

well.

1. Make any needed identifying marks in soft pencil or preferred marker on a piece of the dry,

precut membrane.

Note: Handle the membrane with gloved fingers or smooth-tipped (non-serrated) forceps to

prevent oils and proteins on fingers from blocking efficient transfer.

Note: PVDF membranes require pre-treatment by soaking in methanol for 1 to 2 minutes followed

by soaking in transfer buffer for 5 minutes.

2. Wet the membrane by floating it on distilled water for a few moments and then immerse it in

transfer buffer for 1 to 2 minutes.

3. Align the membrane carefully on the gel taking care not to create any air pockets.

Note: Do not allow the membrane to touch the wicking sheet during transfer. If it does, a ‘short

circuit’will occur and buffer will move around rather than through the gel.

4. Place the tray insert, with the wicking sheet, gel and membrane on it, into the buffer tray with

the ends of the wicking sheets extending into the tray.

5. Place a stack of blotting pads on top of the membrane, and the gel restrainer over the blotting

pads (Figure 5). As with the membrane, be careful not to let the blotting pads touch the wicking

sheet. To ensure even contact through the blotting stack, place a weight on top of the gel

restrainer.

2.4 CAPILLARY TRANSFER

1. Fill the buffer tray approximately half full with transfer buffer. To ensure even capillary flow,

make sure the ends of the wicking sheets are equally immersed.

2. Allow from 4 to 24 hours for complete capillary transfer depending upon the size of the DNA or

RNA fragments and the percentage and thickness of the gel.

Figure 5: Configuration for capillary transfer

Apogee Electrophoresis | 101 Kane Street |Baltimore, MD 21224 USA |apogeephoresis.com

3. After blotting is complete, remove the gel restrainer and blotting pads. Carefully lift the

membrane from the gel with forceps and initiate subsequent analysis procedure.

Note: The gel will normally be compressed to a small fraction of its original thickness.

4. Discard all used paper blotting materials properly. Thoroughly rinse and dry all plastic

components to remove any residual solution.

3.0 REFERENCES

1. Southern, E.M. (1975) Journal of Molecular Biology 98, 503.

2. Maniatis, T., Fritsch, E.F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold

Spring Harbor Laboratory, Cold Spring Harbor, New York.

3. Schatz, D.G. (1989) in Current Protocols in Molecular Biology, (Ausubel, F>M., Brent, R.,

Kingston, R.E., Moore, D.D., Seidman, J.G, Smith, J.A. and Struhl, K., eds.), Vol. 1 p. 2.9.1, Greene

Publishing Associates and Wiley Interscience, New York.

4. Thomas, P.S. (1980) Proceedings National Academy of Acad. Science USA 77, 5201.

5. Selden, R.F. (1989) in Current Protocols in Molecular Biology, (Ausubel, F>M., Brent, R., Kingston,

R.E., Moore, D.D., Seidman, J.G, Smith, J.A. and Struhl, K., eds.), Vol. 1 p. 4.9.1, Greene

Publishing Associates and Wiley Interscience, New York.

6. Bolt & Mahoney, (1997) High-efficiency blotting of proteins of diverse sizes following sodium

dodecyl sulfate–polyacrylamide gel electrophoresis, Analytical Biochemistry 247, 185–192.

4.0 RELATED PRODUCTS

4.1 H11.14 AND H5 GEL SIZE CATALOG #

Horizon 11.14 horizontal gel electrophoresis apparatus 11 x 14 cm gel 11068012

Includes FastCast aluminum wedges and integral safety lid

Model H5 horizontal gel electrophoresis apparatus 11 x 14 cm gel 21087010

H11.14 UVT Tray 11 x 14 cm gel 11084019

4.2 H20.25 AND H4 GEL SIZE CATALOG #

Horizon 20.25 horizontal gel electrophoresis apparatus 20 x 25 cm gel 21069018

Includes FastCast aluminum wedges and integral safety lid

Model H4 horizontal gel electrophoresis apparatus 20 x 25 cm gel 11025012

H20.25 UVT Tray 20 x 25 cm gel 31006026

Apogee Electrophoresis | 101 Kane Street |Baltimore, MD 21224 USA |apogeephoresis.com

5.0 CARE AND HANDLING

5.1 MATERIALS AND CARE

Each apparatus is fabricated from high quality formed PVC/Acrylic and machined acrylic plastic. Both

have very good heat, impact, and chemical resistance but will not withstand autoclaving or dry-heat

sterilization.

All components may be washed with water and a detergent. To remove grease and oils, use a hexane,

kerosene, or aliphatic naphtha. Never use abrasive cleaners, window sprays, or any fluid that may

contain toluene, methylene chloride, phenol, acetone, benzene, halogenated hydrocarbon solvents, or

undiluted laboratory alcohols.

For replacement parts, call your distributor or Apogee Technical Support.

5.2 GENERAL SPECIFICATIONS

Type H11.14 Casting System H20.25 Casting System

Dimensions (W × L x H) 17.6 23.2  cm 26.7 x 34.0 x 5.6 cm

Weight 0.37 kg 0.72 kg

Gel Dimensions 11 x 14 cm 20 x 25 cm

Construction PVC/Acrylic, acrylic PVC/Acrylic, acrylic

5.3 TECHNICAL SUPPORT AND SERVICE

Should you have any problems with this unit, please contact:

Apogee Designs, Ltd.

Attn: Electrophoresis Support

101 Kane Street

Baltimore, MD 21224 USA

Phone: 443.744.0368 9 to 5PM EST, Monday through Friday

Fax: 410.633.3666

Email: info@apogeephoresis.com

5.4 INSTRUCTIONS FOR RETURN SHIPMENT

IMPORTANT: Before sending the unit back to us, it is absolutely necessary to call our Technical

Support department to get authorization to return products!

Return only defective devices. For technical problems which are not definitively recognizable as

device faults please contact Apogee Technical Support.

Apogee Electrophoresis | 101 Kane Street |Baltimore, MD 21224 USA |apogeephoresis.com

Use the original box or a similarly sturdy one.

Please enclose a detailed description of the fault and when, or how, the problem occurred.

Important: Clean all parts from residues and of biologically dangerous, chemical and radioactive

contaminants. Please include a written confirmation (use the respective Decontamination

Declaration/Certificate following in Section 8 that the device is free of biologically dangerous and

radioactive contaminants in each shipment. If the device is contaminated, it is possible that Apogee will

be forced to refuse to accept the device. The sender of the repair order will be held liable for possible

damages resulting from insufficient decontamination of the device.

Please enclose a note which contains the following:

1. Sender's name and address and,

2. Name of a contact person for further inquiries with telephone number.

5.4.1 CLEANING AND DECONTAMINATION FOR RETURN OF PRODUCTS

Use the original product packaging whenever possible, to avoid damage to the unit being returned. All

returned material must be cleaned and decontaminated prior to shipping. The components of

apparatus products are fabricated from a variety of materials including: ABS, acrylic, vinyl, glass, silicone,

aluminum and stainless steel. Please clean any unit or product to be returned using the following three

step procedure.

STEP 1: GENERAL CLEANING PROCEDURE

For materials not contaminated with biological or radiological substances, components may be gently

washed with water and a non-abrasive detergent, and rinsed with deionized water. Dry using a soft

cloth, paper towel or allow to air dry. A light application of hexane, kerosene, or aliphatic naphtha will

remove grease.

To prevent surface damage, never use abrasive cleaners, window sprays or scouring pads to clean these

products. Avoid excessive exposure to UV light, phenol, acetone, benzene, halogenated hydrocarbon

solvents or undiluted alcohols because they may cause crazing.

STEP 2: BIOLOGICAL CLEANING PROCEDURE

Using a solution of either 5% household bleach in water or 70% ethanol in water, wipe down the

apparatus using a clean cloth or sponge. Neutralize the solution by wiping the surface with a mild,

nonabrasive detergent and rinse well with water.

STEP 3: RADIOLOGICAL DECONTAMINATION PROCEDURE

To meet various regulatory and safety standards, please follow the decontamination procedure given

here if radioactive materials are used with this product or are used in the vicinity of where this

apparatus has been used or stored.

WARNING: We cannot and will not accept return of products that are contaminated with any

radioactivity.

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