instructions

GenomiPhi DNAAmplification Kit

i 74004229 Ed. AB

product code:

25-6600-0125-6600-02

WarningFor research use only.Not recommended orintended for the diagnosisof disease in humans or animals.Do not use internally orexternally in humans or animals.

Harmful

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HandlingStorageStore the kit at -70 ˚C.

Note: The enzyme mixmust be stored at -70 ˚C;all other components canbe stored at -20 ˚C.

Thaw components on iceand maintain at 0–4 ˚Cduring handling.

Do not allow any compo-nent to warm above 4 ˚Cprior to the amplificationreaction.

Page finderHandling and storage . . . . . . . . . . . . . . . . . . . . . 3

Components of the kit . . . . . . . . . . . . . . . . . . . . . 4

World Wide Web addresses . . . . . . . . . . . . . . . . . 4

Quality control . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

Safety warnings and precautions . . . . . . . . . . . . . 5

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

ProtocolsOverview1. DNA amplification . . . . . . . . . . . . . . . . . . . . . . 92. Short protocol . . . . . . . . . . . . . . . . . . . . . . . . 143. Control amplification . . . . . . . . . . . . . . . . . . . 144. Quantification of amplified products . . . . . . . 15

AppendixPurification of amplified products. . . . . . . . . . . . 19

Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . 23

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Licenses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

Trademarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

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Components of the kit

GenomiPhi™ DNA Amplification Kit containssufficient reagents to perform 100 amplifica-tion reactions.

Component Quantity Storage

Sample buffer 1 × 0.9 ml -70 ˚C or -20 ˚C(green cap)

Reaction buffer 1 × 0.9 ml -70 ˚C or -20 ˚C(blue cap)

Enzyme mix 1 × 100 µl -70 ˚C(yellow cap)

Control DNA 1 × 20 µl -70 ˚C or -20 ˚C(Lambda, 10 ng/µl)

World Wide Web addresses

http://www.genomiphi.comhttp://www.amershambiosciences.com

Quality controlAmplification (18 h, 30 ˚C)of 10 ng of human genom-ic DNA must produce 4–7 µg of DNA. The qualityand representation ofamplification was deter-mined by real-time PCRusing 5 primer sets specificfor 5 loci on different chro-mosomes. The absence ofhuman-specific sequencein nonspecific amplificaitonproducts was verfified byreal-time PCR.

Materials notsuppliedReagents

• Water—Use onlydeionized, distilledwater.

• Ethanol (100% and70%)—For optionalpost-amplificationcleanup.

Note: Do not use dena-tured alcohol.

Safety warnings and precautions

Warning: For research use only. Notrecommended or intended for diagnosisof disease in humans or animals. Do notuse internally or externally in humansor animals.

Consider all chemicals to be potentiallyhazardous. Only persons trained inlaboratory techniques and familiar withthe principles of good laboratory practiceshould handle this product. Wear suitableprotective clothing such as laboratory over-alls, safety glasses and gloves. Exercisecaution to avoid contact with skin or eyes;if contact should occur, wash immediatelywith water (see Material Safety Data Sheetfor specific recommendations).

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Materials notsupplied continued

Reagents

• Sodium acetate/EDTAbuffer—For optionalpost-amplificationcleanup. Prepare thisbuffer by mixing equalvolumes of 3 M sodiumacetate (pH > 8.0) and0.5 M EDTA (pH 8.0).

Equipment

• Liquid-handlingsupplies—Vials,pipettes, microcen-trifuge, and vacuumcentrifuge. Perform allamplification reactionsin plastic microcen-trifuge tubes (typically0.5 ml), or in 96-well or384-well plates suitablefor sealing and incuba-tion at 30 ˚C.

• Incubator—For incuba-tions at 30 ˚C.

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Introduction

GenomiPhi DNA Amplification Kit amplifies linear genomic DNA innovel fashion (Figure 1). The GenomiPhi method utilizes bacteriophagePhi29 DNA polymerase to exponentially amplify single- ordouble-stranded linear DNA templates during an isothermal (30 ˚C),strand displacement reaction—thermal cycling is not required (1, 2).Microgram quantities of DNA are generated from nanogram amountsof starting material after an overnight incubation. DNA replication isextremely accurate because of the proofreading activity of Phi29 DNApolymerase (3, 4).

The starting material for GenomiPhi reactions can be purified DNA(quantities small as one nanogram) or unpurified cell lysates. Mostcommercial DNA isolation kits and homemade purification proceduresproduce suitable material. Most DNA generated by GenomiPhi is highmolecular weight and double-stranded; a small quantity of single-stranded DNA is produced. Segments within 500 bases of a terminuswill be underrepresented in the amplified sample. GenomiPhi-amplifiedDNA can be used directly in many applications without purification,but can also be purified by suitable methods such as ethanol precipita-tion.

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Random hexamer primers anneal to thetemplate DNA at multiple sites.

Phi29 DNA polymerase initiates replicationat multiple sites on the denatured linearDNA simultaneously.

As polymerization proceeds, strand displace-ment of downstream replicated DNA generatesnew single-stranded DNA.

Additional primers bind to newly synthesizedsingle-stranded DNA.

Subsequent priming and strand displacementproduce large quantities of high-molecularweight, double-stranded DNA.

Fig 1. Schematic diagram of the GenomiPhi amplification process.

primers

template DNA

replicated DNA polymerase

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Protocols

Overview

The kit consists of three components—sample buffer, reaction buffer, andenzyme mix (Phi29 DNA polymerase with random primers). Sample buffercontains random hexamers that nonspecifically prime polymerization cat-alyzed by Phi29 DNA polymerase. The reaction buffer contains salts anddeoxynucleotides, and is adjusted to a pH that is optimal for Phi29 DNApolymerase-catalyzed synthesis.

Briefly, 1 µl of a template to be amplified is added to 9 µl of sample bufferand is heated to 95 ˚C for 3 min to denature the template DNA. The sam-ple is cooled and mixed with 9 µl of reaction buffer and 1 µl of enzymemix, and incubated at 30 ˚C overnight (16–18 h). After amplification, Phi29 DNA polymerase is heat-inactivated during a 10 min incubation at65 ˚C. The tube should now contain amplified DNA that can be useddirectly or after purification in downstream application experiments.

➊ DNA amplification

The steps outlined below describe a general amplification protocol andshould be considered a starting point for optimizing the reaction inyour laboratory. Reaction parameters that can be manipulated for aparticular type of template and laboratory workflow are discussed, asare the method’s limitations.

In the absence of template DNA, an amplification product will likelyappear. Negative control reactions (no template DNA) are expected toproduce an amplification product, but this material will not perform indownstream applications.

Please begin at step 1.1 if you have not optimized your GenomiPhireaction. A short protocol (Protocol 2) is included for researchers whohave optimized their reactions.

1.1 Transfer starting material into sample buffer.

Add 1 µl of the template to be amplified to 9 µl of sample buffer.Sample buffer can be dispensed in 9 µl aliquots into an appropri-ate vessel such as 0.5 ml microcentrifuge tubes, 96-well or 384-well microplates. The vessel must be sealable (i.e. cap) to pre-vent evaporation during subsequent steps, and for storage of theamplified product.

Note: GenomiPhi-compatible preparation methods for varioussamples are provided below. Researchers must verify the suitabil-ity of alternative techniques.

Note: Avoid adding more than 1 µl of template. Contaminantswithin the samples (i.e. SDS, EDTA, certain salts, blood heme) athigh concentration can inhibit Phi29 DNA polymerase.

Note: The kinetics of the GenomiPhi reaction strongly favor intacttemplates. Templates that have been digested with a restrictionenzyme or that are nicked extensively will be poor substrates.Research at Amersham Biosciences indicates that samples from

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formalin-fixed tissues, and templates stored under undesirableconditions for long periods (i.e. old bones, old blood samples),produce poor GenomiPhi amplification.

Purified DNA

Add a small amount (minimum of 1 ng in 1 µl) of DNA dissolvedin water or TE (10 mM Tris, 1 mM EDTA, pH 8.0) into a vesselcontaining 9 µl of sample buffer. Cap the tube or seal the plate.Proceed to step 1.2.

Blood cells

Dilute blood three-fold or more with physiological buffer(e.g. PBS). Mix well and add one part of this mixture to one partof alkaline lysis solution (e.g. 400 mM KOH, 100 mM DTT, 10mM EDTA). Mix well with gentle tapping and incubate on ice for10 min. After incubation, add one part of a neutralization solution(e.g. 400 mM HCl, 600 mM Tris HCl, pH 0.6 prepared by mixing4 ml of 1 M HCl and 6 ml of 1 M Tris HCl, pH 7.5) to the prepa-ration and mix well. Add up to 1 µl of this final lysate into a vesselcontaining 9 µl of sample buffer. Cap the tube or seal the plate.Proceed to step 1.2.

Buccal swabs (cheek cells)

Place the head of a swab (cotton or polyester) in an appropriatecontainer such as a microcentrifuge tube. Add sufficient alkalinelysis solution (described above for blood cells) to submerge theswab completely. Gently vortex briefly and incubate on ice for10 min. Next, add an identical volume of neutralization solution,and mix gently. Transfer up to 1 µl of this final lysate to a freshvessel containing 9 µl of sample buffer. Cap the tube or seal theplate. Proceed to step 1.2.

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Blood blotted paper (e.g. Guthrie card)

Cut the paper into small pieces (3 mm × 3 mm squares).Exercise caution to prevent cross-contamination when the sameapparatus is used to cut multiple samples. Place each piece intoan appropriate container, such as a microcentrifuge tube. Addsufficient alkaline lysis solution (described above for blood cells)to submerge the paper completely. Gently vortex briefly andincubate on ice for 10 min. Next, add an identical volume ofneutralization solution, and mix gently. Transfer up to 1 µl of thisfinal lysate to a fresh vessel containing 9 µl of sample buffer. Capthe tube or seal the plate. Proceed to step 1.2.

Paper blotted with buccal swab (e.g. Guthrie card)

Collect cheek cells with an appropriate swab, and blot the sampleonto paper as quickly as possible. Dry the paper completely, andcut it into small pieces (3 mm × 3 mm squares). Exercise cautionto prevent cross-contamination when the same apparatus is usedto cut multiple samples. Place each piece into an appropriatecontainer, such as a microcentrifuge tube. Add sufficient alkalinelysis solution (described above for blood cells) to submerge thepaper completely. Gently vortex briefly and incubate on ice for10 min. Next, add an identical volume of neutralization solution,and mix gently. Transfer up to 1 µl of this final lysate to a freshvessel containing 9 µl of sample buffer. Cap the tube or seal theplate. Proceed to step 1.2.

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1.2 Denature the sample

Heat the sealed plate or tube for 3 min at 95 ˚C. Cool to 4 ˚C onice. Incubation at higher temperatures or for longer periods cannick the template DNA, decreasing the efficiency of amplification.

1.3 Prepare the GenomiPhi Kit reaction premix

For each amplification reaction, combine on ice 9 µl of reactionbuffer and 1 µl of enzyme mix; add the entire 10 µl volume to thedenatured sample and mix gently. When performing multiplereactions, it is most convenient to prepare a master mix (9 µl ofreaction buffer and 1 µl of enzyme for each template), and use10 µl for each denatured sample.

Note: Reaction buffer and enzyme mix should be combined onice only in sufficient quantity and immediately prior to addition tothe sample. This combined mix contains Phi29 DNA polymerase,reaction buffer, hexamers, and nucleotides—all the componentssufficient to generate large quantities of DNA. Incubation of thismix alone will generate nonspecific DNA. Discard any unusedportion.

1.4 Incubate at 30 ˚C

Seal the tube or plate and incubate at 30 ˚C for 16–18 h.

Note: This kit is designed to generate maximum amplificationwithin 16 hours at 30 ˚C. Incubation may be continued at 30 ˚Cfor up to 24 h with no adverse effect on the amplified DNA;amplification ceases when the available nucleotides are con-sumed. Because the reaction kinetics are exponential, incubationperiods shorter than 6 hours might produce inconsistent yieldsfrom sample to sample.

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1.5 Heat-inactivate the enzyme

Heat the sample for 10 min at 65 ˚C. Cool to 4 ˚C.

This step inactivates the polymerase and exonuclease activitiesof Phi29 DNA polymerase. Carryover polymerase activity caninterfere with subsequent experiments, while exonuclease activitycan damage DNA during long-term storage.

The DNA can now be used in experiments, or stored (4 ˚C or-20 ˚C).

DNA produced by GenomiPhi Kit is of high molecular weight andconcentrated and therefore is somewhat viscous. If the product isdifficult to pipette, dilute it with water or TE prior to use.

1.6 (Optional) Purify amplification products

Some applications are sensitive to residual components of theamplification reaction or carryover from the starting samples.Nonmultiplexed PCR and certain single nucleotide polymorphism(SNP) detection technologies can utilize GenomiPhi-amplifiedDNA without further processing. The need to purify DNA destinedfor multiplex PCR is best determined empirically. As described inupcoming application notes, purification is one option to optimizeyour results. Please refer to the Appendix for two methods thatremove 99% of residual deoxynucleotide and primer from theamplification reaction. The utility of other purification methodmust be verified by the researcher.

The publication of application notes will be announced athttp://www.genomiphi.com. You also can reserve a copy of thisdocument by contacting your local Amersham Biosciences office.

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➋ Short protocol

2.1 Heat denaturation of template in sample buffer

Mix 1 µl of template with 9 µl of sample buffer.Heat to 95 ˚C for 3 min.Cool to 4 ˚C on ice.

2.2 Preparation of the amplification reaction

For each amplification reaction, combine 9 µl of reaction bufferwith 1 µl of enzyme mix on ice.Add this to the cooled sample.

2.3 Incubation at 30 ˚C

Incubate the sample at 30 ˚C for 16–18 h.

2.4 Post-amplification heat inactivation

Heat the sample at 65 ˚C for 10 min.Cool to 4 ˚C.

2.5 Optional purification of amplified products

Purify amplified material if necessary.Two recommended methods are described in the Appendix.

➌ Control amplification

The control DNA in the kit contains 20 µl of lambda DNA at a concen-tration of 10 ng/µl. To perform the control reaction, transfer 1 µl(10 ng) to a vessel that contains 9 µl of sample buffer. Proceed withsteps 1.2–1.5. The control reaction should generate greater than 4 µgof product within 16 h at 30 ˚C.

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➍ Quantification of amplified products

The amount of GenomiPhi Kit-amplified products in the reaction canbe determined with or without purification. If product is not purified,the PicoGreen™ dsDNA Quantitation Assay (Molecular Probes, Inc.#P-7589) is recommended because this method produces datasimilar to values determined by radioisotope incorporation.Quantitation of unpurified amplified products by spectrophotometricabsorption will generate inaccurate results due to the presence of pro-tein and other kit components. Amplified products that have beenpurified can be quantified by either absorption (OD260) or PicoGreenAssay, using the protocols described below.

Note: Quantification of amplified products is reproducible when usingeither PicoGreen or absorbance methods. However, yields calculatedby these two methods can differ as much as 20%.

Option A: UV absorption (for purified amplification products only)

4.1(a) Set the absorption baseline of the spectrophotometer with TEor water. If the amplification product was precipitated, use theresuspending solution to set the baseline.

4.2(a) Any size cuvette may be used to determine absorption.However, small volume micro-cells (< 500 µl maximumvolume) are more desirable since there is less sample loss.Follow the manufacturer's recommendations for the correctuse of micro-cells and spectrophotometer.

4.3(a) If using a microplate absorption reader, generate a DNA stan-dard curve (including blank) to determine the amount ofamplified product in the sample.

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Option B: PicoGreen assay: multi-well plate format

Note: For quantification by PicoGreen assay using a cuvette format,follow the manufacturer's guidelines. Reagent dilution will depend onthe sensitivity of the fluorescence detection instrument. Consult theinstrument manufacturer’s documentation.

Note: Follow the guidelines in the product booklet for additionalinformation regarding storage and handling of PicoGreen AssayKit components.

4.1(b) Prepare TE buffer:

Prepare a 1× TE solution by diluting 20-fold the concentratedTE buffer included in the kit. Use only sterile, distilled, DNase-free water when preparing the dilution. This TE buffer will beused throughout the assay.

4.2(b) Prepare a 1:25 dilution of PicoGreen reagent:

Determine the required volume of a 1:25 dilution ofPicoGreen reagent.

Volume of 1:25 dilution = 100 µl/sample × number of samples

Example: 100 samples require 10 ml of the 1:25 dilution

Determine the volume of stock (undiluted) PicroGreen reagentnecessary to produce the required volume of a 1:25 dilution.

volume of required 1:25 dilution

25

10 ml of 1:25 dilution

25

Important: Prepare this solution in a plastic vessel rather thanglass because the reagent may adsorb to glass surfaces.Protect the solution from light at all times—cover with foil orstore it in the dark.

= 0.4 ml

Volume of stock PicroGreen =

Example:

4.3(b) Prepare DNA to generate a standard curve. Analyze duplicatesamples of the following dilutions to produce a reliable curve.

Dilute the λ DNA standard (supplied in the kit) ten-fold from a10 ng/µl working solution. Use this dilution for the preparationof the assay standards (see below). Add 100 µl of each dilu-tion to each well of the assay plate.

Standard # λ DNA (ng) λ DNA [10 ng/µl] 1× TE

1 600 60 µl 40 µl2 500 50 µl 50 µl3 400 40 µl 60 µl4 200 20 µl 80 µl5 100 10 µl 90 µl6 50 5 µl 95 µl7 25 2.5 µl 97.5 µl8 0 0 µl 100 µl

4.4(b) Add the GenomiPhi-amplified products:

Aliquot 99 µl of TE into each sample well. Add 1 µl of non-diluted sample for a final volume of 100 µl.

Note: Alternatively, dilute the amplified material with TEbefore adding the samples to the PicoGreen Assay. This dilu-tion factor must be taken into consideration when calculatingtotal yields.

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4.5(b) Perform the PicoGreen assay:

1) Add 100 µl of the 1:25 dilution of PicoGreen to all wellscontaining standards and samples. Mix the contents wellby pipetting up and down.

2) Seal the plate and spin it in a microplate centrifuge for1 min at < 200 × g to eliminate bubbles.

Note: Protect the plate from light at all times. The platemust be read 5–10 min after addition of PicoGreen reagent.

3) Measure the sample fluorescence using a fluorescencemicroplate reader. Set the fluorescence reader at an exci-tation wavelength of ~ 480 nm and emission wavelength of~ 520 nm. Set the instrument gain to optimal or in such away that the sample with the highest DNA concentrationgenerates readings that fall within the linear dynamicrange of the instrument.

4) Correct the data by subtracting the value obtained for theblank from that of each of the samples. Generate a stan-dard curve of fluorescence versus DNA concentrationusing the corrected data. Determine the concentration ofGenomiPhi Kit amplified products from the equation of theline derived from the standard curve.

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Appendix:Purification of amplification products

A. Alcohol precipitation

Note: Post-amplification cleanup is most easily and inexpensivelyachieved by ethanol precipitation using the sodium acetate/EDTAbuffer. This buffer contains 1.5 M sodium acetate (pH > 8.0) and250 mM EDTA. Perform these steps at room temperature.

1) After completing protocol step 1.5, cool the reaction toroom temperature.

2) Add 20 µl (same volume as the amplification reaction) of water toeach tube or well.

3) Add 4 µl (1/10 volume) of sodium acetate/EDTA buffer to eachreaction and mix well.

Note: Do not combine the sodium acetate/EDTA buffer and ethanolprior to their addition to the amplified DNA—EDTA has very lowsolubility in ethanol and will precipitate.

4) Add 100 µl of 100% ethanol to each reaction and mix well. Thefinal concentration of ethanol should be 70%.

Note: Chilling the mix to 4 ˚C or lower or increasing the finalethanol concentration by 5% can adversely affect the performanceof this cleanup protocol. Overnight incubation after adding ethanolis acceptable.

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5) Centrifuge the tubes at room temperature in a microcentrifuge for15 min at ~ 12 000 rpm. Centrifuge 96-well plates for at least 30 min at 2 500 × g or greater.

6) Remove the supernatant by aspiration from each microcentrifugetube. For plates, a brief inverted spin (< 1 min at 300 × g) is suffi-cient for supernatant removal. Remove as much liquid as possible.

7) Wash the DNA pellets with 70% ethanol. Use as large a volume of70% ethanol as the tube or well can safely accommodate.Centrifuge briefly at room temperature at > 3 200 × g for 5 min forplates or in a microcentrifuge for 1 min at ~ 12 000 rpm.

Note: Scientists at Amersham Biosciences routinely use 250–500 µlfor 0.5-ml microcentrifuge tubes and 100 µl for 96-well plates.

8) Remove the supernatants by aspiration or by an inverted spin.Air-dry (preferably) or vacuum-dry (in a vacuum centrifuge) theDNA for 2–5 min. Do not overdry—pellets will become difficultto resuspend.

9) Resuspend the pellet in an appropriate amount of TE.

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B. Spin column chromatography

The method described below is used with MicroSpin™ G-50 Columns(Amersham Biosciences, product code 27-5330-01), which are packedwith Sephadex™ G-50.*

Note: If the reaction volume is < 50 µl, adjust to 50 µl with water orTE buffer. If the volume is > 50 µl, apply a 50 µl aliquot to a singlecolumn or use more than one column for the purification.

Note: The flowthrough from these columns may affect spectrophoto-metric absorption readings. If the product will be quantified by absorp-tion, the column must be washed with TE at least twice before loadingthe sample. To wash the column, add 300 µl of TE buffer to the resinand spin according to the speeds shown below and repeat.

1) Column preparation

a. Resuspend the resin in the column by vortexing.

b. Loosen the cap one-fourth turn and snap off the bottom closure.

c. Place the column in a 1.5 ml screw cap microcentrifuge tube forsupport. Alternatively, cut the cap from a flip-top tube and usethis tube as support.

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*Microspin S-300 (Amersham Biosciences 27-5130-01), AutoSeq™ G-50(27-5340-01) or ProbeQuant™ (27-5335-01). Columns can also be applied,following the instructions included with these products.

d. Prespin the column for 1 min at 735 × g (e.g. 3 000 rpm in anEppendorf model 5414C variable-speed centrifuge with an18-position fixed-angle rotor)*. Start the timer and microcen-trifuge simultaneously.

Note: Use columns immediately after preparation to avoid dryingout of the resin.

2) Sample application

a. Place the column in a new 1.5 ml tube and slowly apply 50 µl ofthe sample (diluted if necessary) to the top-center of the resin,being careful not to disturb the resin bed.

b. Spin the column at 735 × g for 2 min. The purified sample is col-lected in the bottom of the support tube.

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*Centrifugation conditions: Before using a MicroSpin G-50 Column, it isimportant to calculate the speed at which the column should be centrifuged.For a force of 735 × g, the appropriate speed can be calculated from the fol-lowing formula:

rpm=(1 000)( 657/r),

where r = radius in mm measured from center of spindle to bottom of rotorbucket and rpm = revolutions per minute. For example, with a rotor havinga radius of 73 mm, the appropriate speed would be 3 000 rpm.

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Troubleshooting

Problem: No amplification.

It is unusual to achieve no amplification. Success of the amplificationreaction can be confirmed by analyzing a small amount of the sample(1 µl) on a low percentage agarose gel (0.6%). The possible causes andsolutions are listed in order of likelihood.

Possible causes/solutions

1. Excessive contaminants carried over from starting materials: Thebest troubleshooting advice is to dilute the sample as much as possi-ble. Components of some biological material such as heme in bloodcan inhibit the GenomiPhi reaction. The GenomiPhi method is verysensitive—only extremely small amounts of input material arerequired. Therefore, the volume of lysate from cells or tissues addedto the GenomiPhi reaction should be as small as possible. If youhave difficulty transferring small volumes, dilute a portion of thesample in PBS buffer or water and add up to 1 µl of the dilutedmaterial. Avoid adding more than 1 µl of sample.

An excellent optimization step is to prepare a dilution series ofstarting material. Add 1 µl of two-fold serially diluted materialand monitor amplification by agarose gel electrophoresis at themost convenient incubation time for your workflow. Perform thecontrol reaction.

2. Low-quality DNA: The kinetics of the GenomiPhi reaction stronglyfavor intact templates. Templates that have been digested withrestriction enzymes or that are severely nicked will be poor sub-strates for GenomiPhi. Avoid template preparation steps that candamage DNA.

3. Inactive enzyme: It is critical that the enzyme be stored properly. Thebest way to ensure this is to store the material at -70 ˚C. If the com-ponents will be consumed within 2 months, -20 ˚C storage may beused. The freezer used must not be a frost-free unit. Perform the con-trol reaction to confirm performance of the kit.

4. Insufficient DNA: Research at Amersham Biosciences demonstratesrobust amplification with very small amounts of template DNA asthe starting material. Perform a broad titration of your startingmaterial to determine the range that produces optimal amplification.

5. Inefficient denaturation: Heating at 95 ˚C for 3 min is sufficient todenature template DNA and facilitate primer annealing. Longerdenaturing times can nick the template and decrease the amplifica-tion efficiency.

Problem: Poor experimental results using GenomiPhi-amplified products

In the absence of input DNA, there will most likely still be an amplifi-cation product. Like any other highly sensitive, exponential amplifica-tion strategy, minute quantities of contaminating DNA can serve as anefficient amplification substrate, but will not perform in the down-stream application reaction. Negative control reactions (no input DNA)are expected to produce an amplification product.

Possible causes/solutions

1. No amplification: Confirm that a product has been generated byanalyzing a small sample (1 µl) on a low percentage agarose gel(0.6%). If no product is observed after staining with a double-stranded DNA-sensitive dyes such as ethidium bromide, see trou-bleshooting above under “Problem: No Amplification.”

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2. Nonspecific amplification, no input DNA: An amplification productis expected even in the absence of template DNA. Titrate your sam-ple to determine the amount that produces optimal amplification ofyour template.

3. Nonspecific amplification, contaminating input DNA: TheGenomiPhi reaction is exquisitely sensitive; minute amounts of anyinput DNA will be efficiently amplified. It is therefore important touse clean implements and containers.

4. Inhibition of reaction by components of the GenomiPhi reaction:For some downstream applications (i.e. multiplex PCR), it is neces-sar to purify GenomiPhi-amplfied DNA. Apply one of the cleanupprocedures recommended in the Appendix.

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References1. Dean, F. et al., Genome Research 11, 1095–1099 (2001).

2. Lizardi, P. et al., Nat. Genet. 19, 225–232 (1998).

3. Estaban, J.A. et al., J. Biol. Chem. 268, 2719–2726 (1993).

4. Nelson, J.R. et al., BioTechniques 32, S44–S47 (2002).

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All goods and services are sold subject to the terms and conditions of sale of the company within theAmersham Biosciences group that supplies them. A copy of these terms and conditions is availableon request.

The GenomiPhi DNA Amplification Kit and the use thereof for DNA synthesis is covered by US patentapplication number 09/920,571 and US patents 5,854,033, 5,198,543, 5,576,204 and 5,001,050 licensedexclusively to Amersham Biosciences Corp.

©Amersham Biosciences Corp 2003—All rights reserved.Printed in the United States.

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Amersham Biosciences UK Limited

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tel 44 0870 606 1921 fax 44 01494 544350

Amersham Biosciences AB

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tel 46 (0) 18 612 00 00 fax 46 (0) 18 612 12 00

Amersham Biosciences Corp

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tel 1-800-526-3593 fax 1-800-329-3593 or 877-295-8102

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tel 49 761 4519-0 fax 49 761 4519 159

Amersham Biosciences (SV) Corp

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tel 1-800-333-5703 fax 1-408-773-8343

TrademarksAmersham and AmershamBiosciences are trade-marks of Amersham plc.

GenomiPhi, Sephadex,AutoSeq, ProbeQuant andMicroSpin are trademarksof Amersham BiosciencesLimited.

PicoGreen is a trademarkof Molecular Probes, Inc.