hybond-n+ amersham ge healthcare

GE Healthcare

AmershamHybond-N+

Product Booklet

Codes RPN82B 82 mm diam, 50 discsRPN87B 87 mm diam, 50 discsRPN132B 132 mm diam, 50 discsRPN137B 137 mm diam, 50 discsRPN1576B 11.5 x 7.3 cm, 50 sheetsRPN119B 11.9 x 7.8 cm, 50 sheetsRPN225B 22.5 x 22.5 cm, 50 sheetsRPN1210B 12 x 10 cm, 20 sheetsRPN1510B 15 x 10 cm, 20 sheetsRPN1520B 15 x 20 cm, 10 sheetsRPN2020B 20 x 20 cm, 10 sheetsRPN2222B 22.2 x 22.2 cm, 10 sheetsRPN3050B 30 x 50 cm, 5 sheetsRPN2250B 22.2 x 22.2 cm, 50 sheetsRPN203B 20 cm x 3 m, 1 rollRPN303B 30 cm x 3 m, 1 rollRPN1782B 82 mm diam, 50 gridded discsRPN1787B 87 mm diam, 50 gridded discsRPN1732B 132 mm diam, 50 gridded discsRPN1737B 137 mm diam, 50 gridded discs

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Page finder1. Legal 3

2. Handling 42.1. Safety warnings and precautions 42.2 Storage conditions 42.3. Stability 4

3. Components 63.1. Main components 63.2 Critical parameters 63.3. Other materials required 7

4. Description 84.1.Quality control 84.2. Solutions 9

5. Protocols 135.1. Protocol for capillary blotting 135.2. Southern blotting - neutral transfer gel treatment

protocol 165.3. Northern blotting - gel preparation and treatment 175.4. Colony and plaque lifts 195.5. Protocol for dot blotting (manual) 235.6. Hybridization in bags and boxes 255.7. Hybridization in tubes 305.8. Stripping protocol - hot SDS procedure 35

6. Additional information 366.1. Determination of the optimum UV crosslinking

conditions using a UV transilluminator 366.2. Recommended applications for blotting membranes 38

7. References 39

8. Related products 40

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1. Legal GE and GE monogram are trademarks of General Electric Company.

AlkPhos Direct, ECF, ECL, Ficoll, Gene Images, Hybond, Hyperfilm,Megaprime, Rapid-hyb, Ready-To-Go, Rediprime, Redivue, Storm andTyphoon are trademarks of GE Healthcare companies.

Alkphos Direct labelling reagents are covered by European patentnumber 120376B1 and sold under licence from European MolecularBiology Laboratory.

ECF substrate is manufactured for GE Healthcare by JBL ScientificInc and is sold under licence from JBL Scientific Inc.

Gene Images hybridization buffer is covered by US patent nuber5512436 and foreign equivalents.

AnalaR is a trademark of BDH Laboratory Supplies.

Gilson and Pipetman are trademarks of Gilson Inc.

SaranWrap is a trademark of Dow Chemical Company.

Trizma is a trademark of Sigma-Aldrich.

© 2006 General Electric Company – All rights reserved.

GE Healthcare reserves the right, subject to any regulatory approval,to make changes in specifications and features shown herein, ordiscontinue the product described at any time without notice orobligation.

Contact your GE Healthcare representative for the most currentinformation and a copy of the terms and conditions.

http://www.gehealthcare.com/lifesciences

GE Healthcare UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK

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2. Handling

2.1. Safety warningsand precautionsWarning: For research useonly. Not recommended or intendedfor diagnosis of disease inhumans or animals. Do not useinternally or externally inhumans or animals.

We recommend that thisproduct is handled only bythose persons who have beentrained in laboratorytechniques and that it is used inaccordance with the principlesof good laboratory practice. Asall chemicals should beconsidered as potentiallyhazardous. It is advisable whenhandling chemical reagents towear suitable protectiveclothing, such as laboratoryoveralls, safety glasses andgloves. Care should be taken toavoid contact with skin or eyes.In case of contact with skin oreyes wash immediately withwater.

Note that the procedure

requires the use of:

Sodium Dodecyl Sulfate: irritant

Formaldehyde: toxic substance

Formamide: toxic substance

Ethidium Bromide: mutagenicsubstance

Sodium Hydroxide: corrosive

Hydrochloric Acid: corrosive

Diethylpyrocarbonate:explosive, toxic substance

This product may also be usedwith radioactive materials.Please follow themanufacturer’s safety datasheet relating to the safehandling and use of thesereagents.

2.2. Storage Membranes should be stored ina clean, dry atmosphere awayfrom excessive heat, light andnoxious fumes. The membranesshould be handled wearinggloves or using blunt endedforceps to preventcontamination.

2.3 StabilityWhen stored under appropriate

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conditions membranes arestable for up to three years.Membranes should be kept inthe bags in which they arereceived. Performance isconsistent when stored underthe recommended conditions.

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3. Components

3.1. MaincomponentsRPN82B82 mm diam, 50 discs

RPN87B87 mm diam, 50 discs



RPN1576B†11.5 x 7.3 cm, 50 sheets† Designed to fit Omni Trayfrom Nalge Nunc International

RPN119B11.9 x 7.8 cm, 50 sheets

RPN225B22.5 x 22.5 cm, 50 sheets

RPN1210B12 x 10 cm, 20 sheets




RPN2222B22.2 x 22.2 cm, 10 sheets


RPN2250B22.2 x 22.2 cm, 50 sheets

RPN203B20 cm x 3 m, 1 roll

RPN303B30 cm x 3 m, 1 roll

RPN1782B82 mm diam, 50 gridded discs




3.2. Criticalparameters• Storage:

Membranes should be storedin a clean, dry atmosphereaway from excessive heat,light and noxious fumes.

• Handling:The membranes should behandled wearing gloves or

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using blunt ended forceps toprevent contamination. Allmembranes should be cutusing clean sharp scissors toavoid damage to themembrane edges.

• Wettability:The wettability of themembranes is important inachieving a consistentperformance. Nylonmembranes are hydrophilicand do not require pre-wetting before use in blottingprocedures. Wetting ishowever advised for largeblots (>100cm2) or whenmultiple blots are hybridizedtogether. Wet the membranefirst in water then equilibratein an appropriate buffer.

• Fixation:The fixation procedure cansignificantly affect theeventual sensitivity of asystem. Sub optimal fixationreduces the amount ofavailable target sequences,particularly followingstripping. Nucleic acid may befixed using heat or UVcrosslinking. It is essential that

fixation times, energy settings(where appropriate) andconcentration (whereappropriate) are optimized.

3.3. Other materialsrequiredEquipment• Agarose gel electrophoresis

apparatus, for example HE33Mini or HE99x Max submarinegel electrophoresis systems

• Microwave• Hybond Blotting Paper• Absorbent paper towels • Trays/dishes • Glass plates • 750 g weight• Pipettes, for example,

Gilson™Pipetman™ P20,P200, P1000 and P5000

• Assorted laboratoryglassware

• Oven, at 80°C or UVtransilluminator

• Orbital shaker• SaranWrap™ or similar cling

film

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4. DescriptionAll Hybond™ membranes are identical on both sides.

There are two distinct manufacturing methods, resulting inmembranes with different characteristics.

a. Unsupported, where the active substrate is cast as a pure sheet:Hybond ECL™. Due to their fragile nature, unsupported membranesshould be handled with care.

b. Supported, where the active substrate is cast onto an inert 'web'or support. Hybond-C extra, Hybond-NX, Hybond-N and Hybond-N+all fall into this class.

Supported nylon membranes have a high binding capacity fornucleic acid, in addition to high tensile strength. For applicationsrequiring a high degree of sensitivity and/or reprobing these types ofmembranes are an ideal choice. Due to its high protein bindingcapacity, nylon requires extensive blocking prior to detection withantibodies to avoid high backgrounds. Consequently, nylonmembranes are not recommended for use in Western blotting.

The membrane, which is identical on both sides, is manufactured inlong rolls known as 'master rolls'. Production runs are carefullycontrolled and constantly monitored to ensure the most consistentproduct reaches the user. Samples are taken from the beginning,middle and end of each master roll and used in single copy genedetection (see Quality Control below).

4.1. Quality controlEvery lot of Hybond-N+ is tested using related GE Healthcareproducts and protocols to ensure maximum compatibility andoptimum performance.

Description: Nylon hybridization transfer membrane

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Application test: Hind III restricted human genomic DNA,separated using neutral agarose gelelectrophoresis, is Southern(1) blotted ontoHybond-N+ and hybridized with N-ras proto-oncogene probe.

Specifications: Detection of 0.5 pg of target DNA. Hybridizationvolume 125 µl/cm2.

Labelling and Performed using the appropriate system:detection: appropriate system:

Megaprime™ random prime labelling kit withRedivue™ [a-32P]-dCTP label, radioactive signaldetected using Hyperfilm™MP

Storage : Store in a clean dry environment.

4.2. SolutionsAll reagents should be of AnalaR™ grade where possible.

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10x nucleic acid loading dyemix

50x TAE (DNA electrophoresisbuffer)

40 mg Bromophenol blue40 mg Xylene cyanol2.5 g Ficoll™ 400Add approximately 8 ml ofdistilled water. Mix to dissolve.Make up to a final volume of 10 ml. Store at roomtemperature for up to 3months.

242 g Trizma™ base18.6 g Ethylenediaminetetra-acetic acid (EDTA), sodium saltAdd approximately 800 ml ofdistilled water. Mix to dissolve.

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50x TAE (DNA electrophoresisbuffer) continued

Depurination solution

Denaturation buffer

Neutralization buffer

Nucleic acid transfer buffer(20x SSC)

Adjust to pH 8 with glacialacetic acid (~57 ml/l). Make upto a final volume of 1000 ml.Store at room temperature forup to 3 months.

11 ml HCI989 ml Distilled waterMix. Store at room temperaturefor up to 1 month.

87.66 g NaCl20 g NaOHAdd approximately 800 ml ofdistilled water. Mix to dissolve.Make up to a final volume of1000 ml. Store at roomtemperature for up to 3 months.

87.66 g NaCl60.5 g Trizma baseAdd approximately 800 ml ofdistilled water. Mix to dissolve.Adjust to pH 7.5 withconcentrated hydrochloric acid.Make up to a final volume of1000 ml. Store at roomtemperature for up to 3 months.

88.23 g Tri-sodium citrate 175.32 g NaClAdd approximately 800 ml of

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Nucleic acid transfer buffer(20x SSC) continued

TE buffer

10x MOPS buffer

100x Denhardt’s solution

distilled water. Mix to dissolve.Check the pH is 7–8. Make up toa final volume of 1000 ml. Storeat room temperature for up to3 months.

1.21 g Trizma base0.372 g EDTA, sodium saltAdd approximately 800 ml ofdistilled water. Mix to dissolve.Adjust to pH 8 withconcentrated hydrochloric acid.Make up to a final volume of1000 ml. Store at roomtemperature for up to 3 months.

41.2 g 3-(N-morpholino)propanesulfonic acid (MOPS) 10.9 g Sodium Acetate, 3-hydrate 3.7 g EDTA, sodium saltAdd approximately 800 ml ofnuclease free distilled water.Mix to dissolve. Adjust to pH 7with NaOH (prepared innuclease free distilled water).Make up to a final volume of1000 ml. Filter sterilize. Store atroom temperature protectedfrom light. Do not use if thesolution appears yellow in color.

2.0 g Bovine serum albumin

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100x Denhardt’s solutioncontinued

2.0 g Ficoll 4002.0 g PolyvinylpyrrolidoneAdd approximately 50 ml ofdistilled water. Mix to dissolve.Make up to a final volume of100 ml. Store at -15°C to -30°Cfor up to 3 months.

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Protocol

5.1.1. Prepare the gel fortransfer.

5.1.2. Cut a sheet of membraneto an appropriate size.

5.1.3. Half fill a tray or glass dishof a suitable size with the transferbuffer. Make a platform and coverwith a wick made from threesheets of Hybond Blotting papersaturated in transfer buffer.

5.1.4. Place the treated gel onthe wick platform. Avoid trappingany air bubbles between the geland the wick. Surround the gelwith cling film to prevent the

Notes

5.1.1. Details of gel treatmentsmay be found on page 16–17,Southern blotting or page17–19, Northern blotting.

5.1.2. The membrane should becut with clean scissors.

5.1.3. At least 800 ml of bufferis required for a 20 x 20 cm geland a dish 24 x 24 cm. Ensurethe wick ends are immersed inthe transfer buffer.

5.1.4. Air bubbles block thetransfer of nucleic acid to themembrane. They may beremoved at any stage by rollinga clean pipette or glass rod

5. ProtocolsThis pack leaflet is limited to the classical capillary blottingtechnique (5.1–5.4) used for the transfer of separated nucleic acidfragments from an agarose gel to a solid support, and isrepresentative of the procedures used in GE Healthcare laboratories.

Details of the gel treatments required before the transfer of DNA or RNAmay be found on pages 12–16. Figure 1 (page 15) is a diagrammaticrepresentation of the transfer apparatus used in this technique.

5.1. Protocol for capillary blotting

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Protocol

transfer buffer being absorbeddirectly into the paper towels.

5.1.5. Place the membrane ontop of the gel. Avoid trappingany air bubbles.

5.1.6. Place three sheets ofHybond Blotting paper cut tosize and saturated in transferbuffer, on top of the membrane.Avoid trapping any air bubbles.

5.1.7. Place a stack of absorbenttowels on top of the HybondBlotting paper at least 5 cm high.

5.1.8. Finally, place a glassplate and a weight on top ofthe paper stack. Allow thetransfer to proceed overnight.The weight should not exceed750 g for a 20 x 20 cm gel.

5.1.9. After blotting, carefullydismantle the transferapparatus. Before separatingthe gel and membrane, markthe membrane to allowidentification of the tracks witha pencil or chinagraph pen.

Notes

over the surface.

5.1.5. Do not attempt to movethe membrane once it hastouched the gel surface.

5.1.8. Small fragments (0.5–1.5 Kb) are rapidlytransferred upwards in a fewhours, larger fragments (>10 Kb) require at leastovernight transfer. Theefficiency of transfer of theselarger fragments can beimproved by depurination.

5.1.9. Rinsing the membranefollowing transfer is notadvised. Extensiveexperimentation at GEHealthcare has shown thatrinsing the membrane beforefixation produces blots ofvariable quality because

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Protocol

5.1.10. Fix the nucleic acid tothe membrane by baking at 80°C for 2 hours or by using anoptimized UV crosslinkingprocedure.

5.1.11. Blots may be usedimmediately. Blots must bethoroughly dried if storage isrequired.

Notes

nucleic acid is removed fromthe membrane during this step.

5.1.10. Details of anoptimization procedure aregiven on page 36. The (UVC 500crosslinker, available from GEHealthcare, has a pre-set UVexposure (70 000 micro-joules/cm2) which is suitable forHybond-N+.

5.1.11. Blots may be rinsed in 2 x SSC before storage orhybridization. Blots should bestored wrapped in SaranWrapdesiccated at roomtemperature under vacuum.

Fig 1. Diagrammatic representation of a capillary blotting apparatus.

Weight

Glass plate

Plastic tray

3 sheets filter paper

Hybond membrane

Gel

Paper towels

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Protocol

5.2.1. Separate the DNAsamples on a suitable neutralagarose gel.

5.2.2. Following electrophoresisvisualize the DNA samples inthe gel with UV light andphotograph.

5.2.3. Process the gel forblotting, between each steprinse the gel in distilled water.

5.2.3a. Depurination Place in0.125 M HCl so that the gel iscompletely covered in thesolution. Agitate gently forapproximately 10 minutes.During this time thebromophenol blue dye presentin the samples will changecolor.

Notes

5.2.1. Efficient separation of arange of DNA fragments maybe achieved by varying thetype and concentration of theagarose in the gel. Ensure theoptimum DNA concentration fordetection is loaded into eachtrack. 0.1 µg/ml EthidiumBromide should be included inthe gel for visualization.

5.2.2. Minimize the exposure ofthe gel to UV light as this maycause excessive nicking of thenucleic acid.

5.2.3a. Depurination is notrequired for DNA fragments <10 Kb in size. Do not overdepurinate, 10 minutes (or untilthe bromophenol blue turnsyellow) is usually sufficient formost samples.

5.2 Southern blotting – Neutral transfer geltreatment protocol

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Protocol

5.2.3b. DenaturationSubmerge the gel in sufficientdenaturation buffer. Incubatefor 30 minutes with gentleagitation. During this time thebromophenol blue dye willreturn to its original color.

5.2.3c. Neutralization Place the gel in sufficientneutralization buffer tosubmerge the gel. Incubate for30 minutes with gentleagitation.

5.2.4. Set up the capillary blotas described on page 13.

5.3. Northern blotting – gel preparation andtreatmentRNA is separated under denaturing conditions, the principle systemscurrently in use are the glyoxal/dimethylsulphoxide and theformaldehyde/formamide procedures. This booklet restricts itself tothe latter. Successful Northern analysis(7,8) depends on the qualityof the reagents used as well as having pure undegraded RNAsamples.

Avoid any contamination with RNases, use sterile disposable plasticswherever possible. Glassware may be treated by baking at 180°Covernight or incubating in 0.2%(v/v) diethylpyrocarbonate (DEPC)followed by autoclaving or baking. Some plastics are also suitablefor DEPC treatment.

Notes

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Notes

5.3.1. The agarose gel is 0.7 Mwith respect to formaldehydeand lx with respect to the MOPSbuffer. This formulation can bescaled up or down asappropriate for the size of gelrequired.

SybrGreen™ or EthidiumBromide (0.01 µg/ml) may beincluded in the gel forvisualization. RNA does notstain as well as the sameamount of DNA with EthidiumBromide. Excessive amounts ofEthidium Bromide will alsoinhibit RNA transfer(9).

5.3.2. Sample must bedeproteinized. Samples may bestored at -15°C to -30°C forshort periods.

Nucleic acid loading buffermust be prepared using RNasefree reagents/solutions.

Protocol

5.3.1. Prepare theMOPS/formaldehyde gel asfollows:Preheat 17.5 ml offormaldehyde and 30 ml 10 xMOPS buffer at 55°C.

Dissolve 3–4.5 g of agarose in250 ml of nuclease free water.Cool to 55°C. Add the 10 xMOPS buffer and formaldehyde.Cast the gel in an appropriateenclosure and allow the gel toset.

5.3.2. Prepare the RNAsample(s), using the table below:

Volume final

(µl) conc

RNA V

Formaldehyde 5.5 2.2M

Formamide 15 50%

10x MOPS buffer 1.5 0.5x

Water 8-V

TOTAL 30

Place the sample(s) at 55°C for15 minutes to denature. Afterdenaturation add 3 µl of 10 x

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Notes

5.3.4 The integrity of the RNAmay be assessed by theabsence of smearing and thefluorescent signal, the ratio of28S to 18S RNA should be 2:1.

5.3.7. 10 x SSC or 20 x SCC canbe used as the transfer buffer.

Protocol

nucleic acid dye loading buffer.Mix and load onto the agarosegel.

5.3.3. Separate the RNAsamples using 1 x MOPS bufferas the electrophoresis buffer.

5.3.4 Following electrophoresis,if appropriate, visualize the RNAwithin the gel with UV light andphotograph.

5.3.5. Place the gel in a suitabletray or dish and cover withdistilled water. Incubate the gelwith gentle agitation for 15minutes.

5.3.6. Discard the water andreplace with sterile 10 x SSC.Agitate for 15 minutes. Repeatthis step once more.

5.3.7. Set up the capillary blotas described on page 10 using aneutral transfer buffer.

5.4. Colony and plaque lifts(10)

Protocol

5.4.1. Plate out the cells or bacteriophage in the usualway. Incubate overnight at the

Notes

5.4.1. Do not allow the coloniesto grow too large. Acolony/plaque density of up to

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Protocol

required temperature.

5.4.2. Pre-cool the petri-dishesfor at least 30 minutes at 2–8°C before taking the lift .

5.4.3. Select the correct size ofmembrane disc.

5.4.4. Bend the membrane andplace the resulting troughacross the centre of the petri-dish. Release the trough andallow the membrane to sit onthe surface. Mark the discposition on the plate at severalpositions using a pin to ensure

Notes

200 per 83 mm plate is optimalfor accurate selection ofpositive clones.

5.4.2. Pre-cooling preventssmearing of the colonies andseparation of the top agarlayer. Plates must be free ofexcess moisture.

5.4.3. The hydrophilic nature ofnylon ensures accuratecolony/plaque lifts. If desiredthe membrane may be pre-wetbefore use, for example on anunused agar plate or on a TEbuffer saturated HybondBlotting paper pad. Excessliquid must be removed fromthe membrane beforeproceeding, this is achieved byplacing the disc on a dry sheetof Hybond Blotting paper, seepage 13.

5.4.4. This procedure willprevent air being trapped underthe membrane. Do not forcethe membrane down, as itunrolls, the membrane disc willflatten. Do not attempt to movethe membrane disc once it hastouched the agar surface.

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Protocol

correct orientation of thecolonies/plaques in subsequentmanipulations.

5.4.5. After 30–60 secondsremove the membrane fromthe petri-dish in one continuousmovement using blunt endedforceps. Place colony/plaqueside uppermost on a sheet ofHybond Blotting paper.

5.4.6. The DNA must beliberated from the bacteria orbacteriophage, denatured andthen fixed to the membranefollowing a neutralization step.This is achieved by placing themembrane discs colony/plaqueuppermost on a series of

Notes

5.4.5. Extending the time themembrane remains on thesurface of the agar will causediffusion of thecolonies/plaques. Replicatefilters can be prepared byplacing a fresh membrane discon top of this templatemembrane. Press themembrane firmly togetherusing a replica plating tool,avoid any lateral movement.Mark the replica membrane.Replica filters should then beincubated on fresh agar platesunder appropriate conditionsuntil colonies of 0.5–1 mmdiameter are obtained.

5.4.6. An initial (optional) lysisstep, 10% (w/v) SDS for 1-3minutes may be included in thecolony lift procedure. TheHybond Blotting paper shouldbe moist, though not too wet asthis will cause diffusion of thecolonies/plaques. Timings

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Protocol

solution saturated HybondBlotting paper pads:-a) Denaturation step,denaturation buffer for 2–5minutesb) Neutralization step,neutralization buffer for 3minutes. Repeat this step oncemore.

5.4.7. Finally, vigorously washthe membrane disc in 2x SSC toremove the proteinous debris.

5.4.8. Transfer the disc, DNAside Hybond Blotting paper, airdry.

5.4.9. Fix the DNA to themembrane by baking for 2hours at 80°C or by using anoptimized UV crosslinkingprocedure.

5.4.10. Membranes may be

Notes

should be optimized, prolongedincubations will cause diffusionof the target DNA makingaccurate selection of positiveclones difficult. Avoid fluidreaching the upper surface ofthe membrane. Whentransferring membrane,remove as much fluid aspossible from the underside ofthe membrane. This may beachieved by transferring brieflyto dry Hybond Blotting paperbetween treatments.

5.4.7. Adequate removal of celldebris from colony lifts isessential.

5.4.9. Details of an optimizationprocedure using a UVtransilluminator are given onpage 36. The UVC 500 UVcrosslinker, available from GEHealthcare, has a pre-set UVexposure (70 000 micro-joules/cm2) which is suitable forHybond-N+.

5.4.10. Membranes should be

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5.5. Protocol for dot blotting (manual)The following is a general protocol for dot blotting target nucleicacids. A number of commercially available devices are alsoavailable, for example the PR648 Slot blot manifold available fromGE Healthcare. These provide for a more consistent and evenapplication of the sample than the manual procedure describedbelow. This parameter is particularly important in those experimentsrequiring quantification.

Protocol

5.5.1. Cut the membrane to anappropriate size.

5.6.2. Using a pencil, mark themembrane lightly with a grid ordots to guide subsequentsample application. Thereshould be a minimum distanceof 1 cm between samplesapplied in a volume 5 µl or less.

5.5.3. Pre-wetting themembrane is not required.

5.5.4. Dilute the samples in anappropriate buffer to therequired concentration. TEbuffer or 2x SSC may be usedfor DNA samples. RNA samples

Notes

5.5.1. The membrane should becut with clean scissors.

5.5.3. Membranes may be pre-wet if desired, see criticalparameters page 6.

5.5.4. Carrier substance may beincluded in the diluent buffer toimprove retention of very smallamounts of target on themembrane. These include:

Notes

stored wrapped in SaranWrapdesiccated at roomtemperature under vacuum.

Protocol

used immediately or stored,once dry.

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Protocol

should be prepared using theinformation on pages 17–19. Asample size of 1–2 µl is ideal formanual dot blotting.

5.5.5. Nucleic acid samplesmust be denatured to provide asuitable single-stranded targetmolecule for subsequenthybridizations. Denature thesamples by heating in a boilingwater bath for 5 minutes. Chillrapidly on ice, then centrifugebriefly to collect sample at thebottom of the tube.

5.5.6. Carefully apply thesample to the membrane,avoiding touching themembrane with the pipette tip.Leave the membrane to air dry.

5.5.7. Fix the nucleic acidsample to the membrane by UV

Notes

• sonicated herring sperm DNAfor DNA samples• tRNA for use with RNAsamples.

Larger sample volumes of 50–200 µl are common forcommercial apparatus. Thisensures an even application ofthe sample over the whole dotor slot.

5.5.5. RNA samples may bepreheated to 55°C for 15minutes, see page 18.

5.5.6. If the sample volume isgreater than 2 µl, then apply insuccessive 2 µl aliquots to thesame position on themembrane, allow the aliquot todry between each application.This will prevent the samplespreading.

5.5.7. Details of an optimizationprocedure are given on page

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Protocol

crosslinking or baking at 80°Cfor 2 hours.

5.5.8. Blots may be usedimmediately or stored wrappedin SaranWrap desiccated atroom temperature undervacuum.

Protocol

5.6.1. Prepare the hybridizationbuffer, for exampleDenhardt’s buffer5 x SSC5 x Denhardt’s solution0.5% (w/v) SDS

Modified Church and Gilbertbuffer(11)7% (w/v) SDS

Notes

32. The UVC 500 UV crosslinker ,available from GE Healthcare,has a pre-set UV exposure (70 000 micro-joules/cm2)which is optimum for Hybond-N+.

Notes

5.6.1. There are a wide varietyof hybridization buffers used byresearchers. This Denhardt’sbased buffer is used in thetesting of Hybond nylonmembranes. A reducedconcentration of SDS has beenfound to elevate backgroundsfollowing hybridization. TheDenhardt’s hybridization buffermay be stored at -15°C to -30°C if required.

This modification of the Churchand Gilbert buffer, is routinelyused in GE Healthcare’

5.6. Hybridizations in bags and boxes (5,6)

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Protocol

0.5 M phosphate buffer, pH 7.210 mM EDTA

5.6.2. Prepare the radiolabelledprobe using the appropriateprocedure.

5.6.3. Preheat the requiredvolume of hybridization bufferto the appropriate temperature.

Notes

laboratories. It has been shownto be suitable for Southerns,Northerns, dot blots and libraryscreening applications. Thehybridization buffer may bestored at room temperature.Ensure the SDS is fully dissolvedbefore use. This may beachieved with gentle heating.

5.6.2. For radioactiveapplications use a probeconcentration of 0.5–2 x 106

incorporated counts per ml ofhybridization buffer for singlecopy gene detection, i.e. highsensitivity application or0.125–0.5 x 106 incorporatedcounts per ml of hybridizationbuffer for high target work, forexample colonies/plaques, PCRproducts etc. Probe purification,to remove unincorporatedradioactive nucleotides, isstrongly recommended.

5.6.3. Pre-wetting in a suitablebuffer is essential for large blots(>100cm2) or multiple blots.Details of the pre-wettingprocedures are given on page4, critical parameters.

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Notes

Hybridization may be carriedout in bags, or boxes, providedthere is sufficient buffer for thecontainer. Adequate circulationof the buffer is essential. Whenhybridizing several blotstogether, the blot should movefreely within the buffer.

Protocol

5.6.4. Pre-wet the blot in asuitable buffer for example 5 xSSC or 0.5 M phosphate buffer.Place the blot(s) in thehybridization buffer. 125 µl ofhybridization buffer per cm2 isa suitable volume. Prehybridizefor at least 30 minutes withconstant agitation, at thedesired hybridizationtemperature (see step 7).

5.6.5. When using labelleddouble stranded probes, pipettethe required amount into aclean microcentrifuge tube. Ifthe volume is less than 20 µl,make up to this volume withwater or TE buffer. Denature theprobe by boiling for 5 minutesand snap cool on ice. Brieflycentrifuge to draw the contentsto the bottom of the tube.

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Protocol

5.6.6. Add the probe to the pre-hybridization buffer.

5.6.7. Hybridize overnight withgentle agitation at the requiredtemperature.

5.6.8. Prepare the stringencywash solutions. The washsolution should be used inexcess, at least 1-5 ml/cm2 ofmembrane.

Notes

5.6.6. Avoid placing the probedirectly on the blots, as this willcause excessive background.

5.6.7. Hybridizationtemperatures may vary withthe probe. Lower temperaturesachieve lower stringency. Thetemperature of hybridizationused will depend on the degreeof homology between theprobe and the target. 65–68°Cis suitable for most longprobes(>100 bases). Withshort/oligo probes (<50 bases)hybidization temperature areusually defined as Tm-5°C:

Tm (melting temperature) = (4 xnumber of G+C bases) + (2 xnumber of A+T bases) (12)

Hybridization time can alsovary. Short hybridization timesmay be suitable for high targetapplications.

5.6.8. Stringency washes willdepend on the nature of theprobe and target to behybridized. Salt concentrationand temperature should betaken into consideration. Thelower the salt concentration,

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Protocol

Low stringency wash:2 x SSC, 0.1% (w/v) SDS

Medium stringency wash:1 x SSC, 0.1% (w/v) SDS

High stringency wash:1 x SSC, 0.1% (w/v) SDS

5.6.9. After the hybridization,wash the blots by incubatingtwice, 5 minutes each, in 2 xSSC, 0.1% SDS, followed by 1 xSSC, 0.1% SDS for 15 minutes,and finally 0.1 x SSC, 0.1% SDSfor 2 x 10 minutes, at the

Notes

the greater the stringency. Thehigher the washingtemperature, the greater thestringency.

Most commonly, stringencywashes proceed from 'highsalt/low temperature', forexample 5 x SSC, 0.1% SDS atroom temperature, to 'lowsalt/high temperature', forexample 0.1 x SSC, 0.1% at65°C (nominal hybridizationtemperature). Some proceduresinclude room temperaturewashes under low stringencyconditions. Do not allow theSDS to come out of solutionduring these washes, significantlevels of background mayresult. Adequate circulation ofthe stringency buffer isessential when washing.Washing in boxes is advised.

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5.7. Hybridization in tubesThere are numerous commercially available rotisserie devicessuitable for use as hybridization ovens (for example GE Healthcarehybridization oven/shaker RPN2510E/251E). These canaccommodate 2–7 tubes. The major advantage of this approach tohybridization is the use of low volumes of hybridization buffer, andtherefore minimal probe volumes. This is achieved because fluid isable to move continually over the membrane.

Notes

5.6.10. The use of SaranWrapwith 35S labelled probes willsignificantly increase exposuretimes. In this case the blotshould be air dried beforeautoradiography, if reprobing isnot required.

Protocol

hybridization temperature.

5.6.10. Remove the blot fromthe last stringency wash, drain,wrap in SaranWrap and exposeto X-ray film, for exampleHyperfilm MP. Keep the blotmoist if it is to be reprobed. Ifreprobing is desired, it may bemore suitable to seal the blot ina plastic bag.

Protocol

5.7.1. Prepare the hybridizationbuffer, for example:

Notes

5.7.1. There are a wide varietyof hybridization buffers used byresearchers. This Denhardt’sbased buffer is used in thequality control of all Hybondnylon membranes. A reducedconcentration of SDS has beenfound to elevate backgroundsfollowing hybridization.The Denhardt’s hybridization

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Protocol

Denhardt’s buffer5 x SSC5 x Denhardt’s solution0.5% (w/v) SDS

Modified Church and Gilbertbuffer(13)0.5 M phosphate buffer, pH 7.2 7% (w/v) SDS l0 mM EDTA

5.7.2. Prepare the radiolabelledprobe using the appropriateprocedure.

5.7.3. Preheat the requiredvolume of hybridization bufferto an appropriate temperature.

5.7.4. Pre-wet the blot in asuitable dish, first in water thenin an appropriate buffer. Ensurethat the nucleic acid side isuppermost. Roll the blot alongits length in such a way as tominimize overlap in the tube.Place inside the hybridization

Notes

buffer may be stored at -15°Cto -30°C if required.

This modification of the Churchand Gilbert buffer, is routinelyused in GE HealthcareLaboratories. It has been shownto be suitable for Southerns,Northerns, dot blots and libraryscreening applications. Thehybridization buffer may bestored at room temperature.Ensure the SDS is fully dissolvedbefore use. This may beachieved with gentle heating.

5.7.3. High backgrounds willresult if sub optimum volumesare used for the membrane andhybridization conditions.

5.7.4. If there is significantoverlap of the blot use of anylon mesh should beconsidered. The mesh achievesseparation of the blot layersallowing better probe access tothese areas. It is stronglyadvised that hybridization

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Protocol

tube.

5.7.5. Add a small volume ofappropriate buffer to thehybridization tube, cap thetube. Unroll the blot by rotatingthe tube in the oppositedirection to the 'rolled' blot.

5.7.6. Drain the tube of excessliquid and replace with theappropriate volume ofhybridization buffer.

5.7.7. Prehybridize for 30minutes at the appropriatetemperature. Ensure that thetube is placed in the correctorientation within the oven toavoid 'rolling' up of the blot.

5.7.8. When using labelleddouble stranded probes, pipette

Notes

volume should be increased (70–125 µl/cm2).

The nylon mesh should be atleast 0.5 cm larger than theblot. Place the mesh in the pre-wetting solution before the blot,in subsequent manipulationstreat as 'one'. The nylon meshmay be reused after washing in10% (w/v) SDS and extensiverinsing in distilled water.

5.7.5. It is important not toallow air to become trappedbetween the inner surface ofthe tube and the membrane.This can cause areas of nosignal or background followinghybridization.

5.7.6. High backgrounds willresult if sub-optimum volumesare used for the membrane andhybridization conditions.

5.7.8. For radioactiveapplications, use a probe

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Protocol

the required amount into aclean microcentrifuge tube. Ifthe volume is less than 20 µl,make up to this volume withwater or TE buffer. Denature theprobe by boiling for 5 minutesand snap cool on ice. Brieflycentrifuge to draw the contentsto the bottom of the tube

5.7.9. Add the probe to the pre-hybridization buffer.

5.7.10. Hybridize overnight atthe required hybridizationtemperature.

Prepare the stringency washsolutions. The wash solutionshould be used in excess.Use a volume that occupies33–50% of the tube.

Notes

concentration of 0.5–2 x 106

incorporated counts per ml ofhybridization buffer for singlecopy gene detection, i.e. highsensitivity application or0.125–0.5 x 106, incorporatedcounts per ml of hybridizationbuffer for high target work, forexample colonies/plaques, PCRproducts etc. Probe purification,to remove unincorporatedradioactive nucleotides, isstrongly recommended.

5.7.9. Avoid placing the probedirectly on the blot. Probe maybe added to the hybridizationwhile the tube is in a verticalposition. If necessary probemay be mixed with a portion ofthe hybridization buffer andadded to the tube in a largervolume.

5.7.10. Hybridizationtemperatures may vary withthe probe. Lower temperaturesachieve lower stringency. Thetemperature of hybridizationused will depend on the degreeof homology between theprobe and the target.

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Protocol

Low stringency wash;2 x SSC, 0.1% (w/v) SDS Medium stringency wash:1 x SSC, 0.1% (w/v) SDSHigh stringency wash:0.1 x SSC, 0.1% (w/v) SDS

5.7.11. After the hybridizationwash the blot as follows:a) rinse briefly in 2 x SSC, 0.1%(w/v) SDSb) wash twice, 5 minutes eachin 2 x SSC,0.1% (w/v) SDSc) wash twice, 10 minutes eachin 1 x SSC,0.1% (w/v) SDSd) wash four times, 5 minuteseach in 0.1 x SSC, 0.1% (w/v)SDS

5.7.12. Remove the blot fromthe last stringency wash, drainand wrap in SaranWrap andexpose to X-ray film, forexample Hyperfilm MP. Keepthe blot moist if it is to bereprobed.

Notes

65–68°C is suitable for mostlong probes(>100 bases). Withshort/oligo probes (<50 bases)hybidization temperature areusually defined as Tm-5°C:

Tm (melting temperature) = (4 xnumber of G+C bases) + (2 xnumber of A+T bases) (14)

Hybridization time can alsovary. Short hybridization timesmay be suitable for high targetapplications.

5.7.11. Washing in boxes ismuch more effective and isrecommended if feasible. Theinefficiency of washing in tubesmay be overcome byincreasing the number ofstringency washes whilemaintaining the same totalwash time.

5.7.12. The use of SaranWrapwith 35S labelled probes willsignificantly increase exposuretimes. In this case the blotshould be air dried beforeautoradiography, if reprobing isnot required.

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5.8. Stripping protocol - Hot SDS procedureProtocol

5.8.1. Place the moistmembrane in an appropriatesized tray.

5.8.2. Prepare a boiling solutionof 0.1% (w/v) SDS, pour thesolution onto the blot and allowto cool.

5.8.3. Rinse the blot briefly in 2 x SSC.

5.8.4. Check the removal of theprobe using the appropriateprocedure for the labelling anddetection system used.

5.8.5. Hybridize overnight usingthe appropriate conditions.

Notes

5.8.2. This step may berepeated if the probe isparticularly difficult to remove.

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Protocol

6.1.1. Produce five or sixidentical control blots, forexample Lambda Hind III on themembrane of choice.

6.1.2. Protect the surface of themembrane by covering thetransilluminator withSaranWrap. Expose each blotDNA side down on thetransilluminator for a differentlength of time, for example 30seconds to 5 minutes.

6.1.3. Hybridize all the blots

Notes

6.1.1. The type of blot shouldreflect the technique for whichthe calibration is being used.

6.1.2. The length of exposurerequired for optimum fixationwill vary depending on thewavelength of the UV bulb andits age. The energy emittedfrom a UV bulb is reduced withuse. Regular recalibration isadvised if the apparatus isextensively used. Thisinconvenience may beovercome with the use of UVcrosslinkers which are able tocompensate for this effect,when used on the constantenergy setting. A UV crosslinkerwith pre-set or manual energyand time settings is availablefrom GE Healthcare (the UVC500 UV crosslinker).

6. Additional information

6.1. Determination of the optimum UVcrosslinking conditions using a UVtransilluminator

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Protocol

together with a suitablylabelled probe.

6.1.4. Followingautoradiography, the optimumUV exposure time will beindicated by selecting the blotshowing the strongest signal.

Notes

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Hybond membranes for binding nucleic acidApplications Hybond-NX Hybond-XL Hybond-N+ Hybond-N

(nylon) (positively (positively (neutralcharged charged nylon)nylon) nylon)

Southern blottingDNA fingerprinting + + + ++Radioactive ++ +++ ++ ++ECL - - +++ -AlkPhos Direct‘ - - +++ -Gene Images‘ - - +++ -Alkali blotting/fixation -- +++ -- --Low volume hybridizations +++ +++ + --Rapid-hyb‘buffer + +++ ++ +

Northern blottingRadioactive detection ++ +++ + ++Non-radioactive detection - - ++ -

Dot/slot blotsRadioactive detection ++ +++ ++ ++Non-radioactive detection - - ++ -

Colony/plaque liftsRadioactive detection +++ ++ + ++Non-radioactive detection + - ++ +

Hybond membranes for binding proteinApplications Hybond-P Hybond ECL Hybond-C Extra

Western blottingECLdetection +++ +++ +ECL Plus detection +++ ++ +Chromogenic detection ++ ++ +Colloidal gold detection ++ ++ -ECF™ detection +++ + -Radioactive detection + + ++Glycoprotein detection +++ + +Reprobing Westerns +++ - +Expression screening + - +++

Key: Suitable = +, Recommended = ++, Highly recommended = +++Not recommended = -, Unsuitable = --

38

6.2. Recommended applications for blottingmembranes

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7. References1. Southern, E.M., J. Mol. Biol. 98, 503–517 (1975).

2. Lichenstein, A.V. et al., Anal. Biochem. 191, 187 (1990).

3. Smith, G.E. and Summer, M.D., Anal. Biochem. 109, 123 (1980).

4. Chomczynski, R., Anal. Biochem. 201, 134 (1992).

5. Olszewska, E. and Jones, K., Trends Genet. 4, 92 (1988).

6. Bittner, M.et al., Anal. Biochem. 102, 459 (1980).

7. Alwine, J.C. et al., Proc. Natl. Acad. Sci. USA 74, 5350–5354 (1977).

8. Alwine, J.C. et al., Methods Enzymol. 68, 220 (1979).

9. Thomas, P.S., Methods Enzymol. 100, 255 (1983).

10. Sambrook, J. and Russell, D., in Molecular Cloning, a Laboratorymanual, Third Edition, Cold Spring Harbor Laboratory Press, (2001).

11. Church, G.M. and Gilbert, W., Proc. Natl. Acad. Sci. USA 81, 1991(1984).

12. Wallace, R.B. and Miyada, C.G., Meth. Enzymol. 152, 438 (1990).

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8. Related productsDNA labelling kitsMegaprime DNA Labelling System dNTP 30 reactions RPN1604

AlkPhos Direct Labelling and DetectionSystem with ECF RPN3692

Rediprime‘II DNA Labelling System30 reactions RPN1633

Ready-To-Go™ You-Prime First-Strand Beads 27-9264-01First-Strand cDNA Synthesis Kit 27-9261-01

Gene Images DNA Random-Prime Labelling Kit 30 reactions RPN3520

Gene Images 3’ Oligo Labelling Kit 30 reactions RPN5770

NucleotidesRedivue formulation 32P- and 33P-labelled radionucleotides

Standard formulation 32P- and 33P-labelled radionucleotides

Additional productsRapid-hyb Buffer 125 ml RPN1635

Rapid-hyb Buffer 500 ml RPN1636

Liquid Blocking Reagent 100ml RPN3601

Scanning instrumentationTyphoon‘ 8600 Variable Mode Imager

Storm‘ Gel and Blot Imaging System

40

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Contact your GE Healthcare representative for the most currentinformation.

41

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http://www.gehealthcare.com/lifesciencesGE Healthcare UK LimitedAmersham Place Little Chalfont Buckinghamshire HP7 9NA UK

GE Healthcareregional officecontact numbers:

Asia PacificTel: +85 65 6 275 1830

Fax: +852 2811 5251

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Fax: +61 2 9899 7511

AustriaTel: 01/57606-1619

Fax: 01/57606-1627

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Fax: 02 416 82 06

CanadaTel: 1 800 463 5800

Fax: 1 800 567 1008

Central, East, & SouthEast EuropeTel: +43 1 982 3826

Fax: +43 1 985 8327

DenmarkTel: 45 16 2400

Fax: 45 16 2424

Finland & BalticsTel: +358-(0)9-512 39 40

Fax: +358 (0)9 512 39 439

FranceTel: 01 6935 6700Fax: 01

6941 9677

GE Healthcare offices:

GE Healthcare Bio-Sciences AB

Björkgatan 30 751 84

Uppsala

Sweden

GE Healthcare Europe GmbH

Munzinger Strasse 5 D-79111

Freiburg

Germany

GE Healthcare UK Limited

Amersham Place

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Buckinghamshire

HP7 9NA

UK

GE Healthcare Bio-Sciences Corp

800 Centennial Avenue

P.O. Box 1327

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NJ 08855-1327

USA

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Sanken Bldg. 3-25-1

Hyakunincho Shinjuku-ku

Tokyo 169-0073

Japan

GermanyTel: (089) 96281 660

Fax: (089) 96281 620

ItalyTel: 02 27322 1

Fax: 02 27302 212

JapanTel: +81 3 5331 9336

Fax: +81 3 5331 9370

Latin AmericaTel: +55 11 3933 7300

Fax: + 55 11 3933 7304

Middle East & AfricaTel: +30 210 9600 687

Fax: +30 210 9600 693

NetherlandsTel: 0800 8282821

Fax: 0800 8282824

NorwayTel: 815 65 555

Fax: 815 65 666

PortugalTel: 21 417 7035

Fax: 21 417 3184

Russia & other C.I.S. &N.I.STel: +7 (095) 232 0250

Fax: +7 (095) 230 6377

+7 (095) 956 1137

South East AsiaTel: 60 3 8024 2080

Fax: 60 3 8024 2090

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SpainTel: 93 594 49 50

Fax: 93 594 49 55

SwedenTel: 018 612 1900

Fax: 018 612 1910

SwitzerlandTel: 0848 8028 12

Fax: 0848 8028 13

UKTel: 0800 616928

Fax: 0800 616927

USATel: +1 800 526 3593

Fax: +1 877 295 8102

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