quickgene dna whole blood kit l (db-l) - wako-chem.co.jp · the material safety data sheet for...

HANDBOOK

QuickGene DNA whole blood kit L(DB-L)

For Isolation of Genomic DNA from whole blood

Ver.2.0

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Warning: For research use only. Not recommended and intended for diagnostic or clinical application for human and animals.

Contents1. Introduction.................................................................................................. 32. Kitcomponents............................................................................................ 33. Storageconditions....................................................................................... 34. Otherrequiredmaterials,notsuppliedinthiskit.......................................... 45. Safetywarnings........................................................................................... 56. Precautions.................................................................................................. 67. Qualitycontrols............................................................................................ 68. Protocols...................................................................................................... 7 8-1Preparationofreagents............................................................................. 7 8-2Samplepreparations................................................................................. 8 8-3GenomicDNAisolationusingtheQuickGene-610L.................................... 109. Troubleshooting.......................................................................................... 1210.OrderingInformation.................................................................................. 14Appendix1...................................................................................................... 15

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1. IntroductionQuickGene porous membrane to immobilize nucleic acid has large specific surface area anduniform & fine porousness.SoQuickGene successfully isolates genomic DNA with high yield;moreover,with its patented thinmembrane, it eliminatesmost contaminants.QuickGenealsousespressured filtration technology,which cannot be successfully utilizedwith typical glassmembranes;byusingpressuredfiltrationtechnology;new,compactandautomaticinstrumentsforrapidnucleicacidpurificationcanbeproducedsuccessfully.WhenQuickGeneDNAwholebloodkitLisusedwithAutomaticNucleicAcidIsolationSystems(QuickGene-610L), highquality and high yield genomic DNAcanbe isolated andalso purifiedfromwholeblood. Inaddition,DNA from6setsofwholebloodsamplescanbesimultaneouslyextracted in only 12 minutes.The purified, high quality genomic DNA is suitable for PCR,restrictionenzymedigestion,southernblottingandotherapplications.

Please be sure to read this handbook carefully before using the kit.This Kit is only used with QuickGene-610L.

2. Kit componentsThekitincludesthereagentsnecessaryfor48setsofgenomicDNAisolation.

Protease (EDB) 5tubes LysisBuffer (LDB) 2bottles WashBuffer (WDB) 4bottles ElutionBuffer (CDB) 1bottle Cartridges (CAL2) WasteTubes (WTL)

3. Storage conditionsAllreagentsarestableforoneyearatroomtemperature(15-28°C).Thedissolvedprotease(EDB)willbeabletostorefortwomonthsat4°C.

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4. Other required materials, not supplied in this kit◆ Reagents •>99%Ethanol •Nuclease-freeultrapurewater(fordissolvingproteases)

◆ Instruments and equipments •QuickGene-610L •50mland15mlcentrifugetubes✽

•Micropipettesandtips •1.5mlmicrotubes(forelutioncollection)✽✽ •Vortexmixer •Tubestands •Tabletopwaterbath(forincubationof50mlor15mlcentrifugetubesat56°C) •500mlreagentbottle(forkeepingthecapsofwashbufferbottle)

✽; 15ml (or 50ml) centrifuge tubesareused for samplepreparation. 50ml centrifuge tube isusedforthecontainersforElutionBuffer(CDB)forQuickGene-610L.

Recommendedcentrifugetube;BDFalcon™50ml,15mlconicaltube.✽✽;Recommendedmicro-centrifugetube;Eppendorf™1.5mlMicroStandardtube.

Table1Recommendedcentrifugetubes

Type of centrifuge tube Product name (Examples)

50mlcentrifugetube BDFalcon™50mlconicaltube

15mlcentrifugetube BDFalcon™15mlconicaltube

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5. Safety warningsWarning: Forresearchuseonly. Not recommended and intended for diagnostic or clinical application for human and

animals.

•All reagentsand itemsshouldbeconsideredchemicallyandbiologicallyhazardous.Wearingalaboratorycoat,glovesandsafetyglassesduringtheexperimentsarehighlyrecommended.Incaseofcontactbetweenthereagentsandtheeyes,skin,orclothing,washimmediatelywithwater.(SeetheMaterialSafetyDataSheetforspecificrecommendations,http://www.kurabo.co.jp/bio/English/)

Protease (EDB) Donotputreagentsineyesandbecarefulofaccidentalingestion. Incaseofcontactbetween the reagentsandeyes,skinorclothing,wash immediatelywith

water.

Lysis Buffer (LDB) Poisonous if swallowed Donotputreagentsineyesandbecarefulofaccidentalingestion. Incaseofcontactbetween the reagentsandeyes,skinorclothing,wash immediatelywith

water. Wearlaboratorycoat,glovesandsafetyglassesduringexperiments.

Wash Buffer (WDB) Donotputreagentsineyesandbecarefulofaccidentalingestion. Incaseofcontactbetween the reagentsandeyes,skinorclothing,wash immediatelywith

water.

Elution Buffer (CDB) Donotputreagentsineyesandbecarefulofaccidentalingestion. Incaseofcontactbetween the reagentsandeyes,skinorclothing,wash immediatelywith

water.

•KeepawaytheLysisBuffer(LDB)fromheat.Donotmixwithdisinfectantssuchasbleach.•For disposal of waste fluid and consumables:Whenusingpotentially infectious samples for

experiments,disposethemaccordingtoapplicableregulations.

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6. Precautions•RefertotheMSDS(MaterialSafetyDataSheet) forspecificrecommendationsonpropertiesand

handling.TheMSDScanbeobtained from theWorldWideWebsite (http://www.kurabo.co.jp/bio/English/).

•Refertotheuser’sguidefortheQuickGene-610Lbeforeusing.

7. Quality controls•Thestabilityofthereagentsisguaranteedforoneyearafterpurchaseifstoredatthespecified

temperature(15-28°C).•Aspart of the stringent of quality assurance program inKURABO INDUSTRIES LTD., the

performanceofQuickGeneDNAwholebloodkitLisevaluatedroutinelyonalot-to-lotuniformity.•Quality and yield of isolatedgenomicDNAsare checkedbymeasuring the absorbance at

260nm,ratioofabsorbance(260nm/280nm).

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8. Protocols

8-1 Preparation of reagents

Protease (EDB)Add3.3mlofnuclease-freeultrapurewatertothevialcontainingthefreeze-driedprotease,anddissolveitcarefully.Storethedissolvedprotease(EDB)at4°C.Thedissolvedprotease(EDB)willbeabletostorefortwomonthsat4°C.Theenzymewillbestable fora longerperiodat -20°C.Recommendtoavoidrepeatedfreezingandthawing.

Notice: Usetheprotease(EDB)afterdissolvingitcompletelywiththefollowinginstructions.Add3.3mlofnuclease-freeultrapurewater,andvortexwiththecapclosed.Leave the protease (EDB) solution 30-40 minutes in room temperature and mix it a few times.Makesureifallthepowderinthesolutionisdissolvedcompletelybeforeuse.Ifitisnotdissolvedcompletely,theyieldwouldbeinsufficientorthecartridgeswouldbeclogged.

Lysis Buffer (LDB)Mixthoroughlybeforeusing.IftheprecipitatesarecontainedinLysisBuffer(LDB),incubatethebottleinawaterbathat37°Candmixwithinversionthebottleintermittentlyuntiltheprecipitatesaredissolved.AfterdissolvingtheLysisBuffer(LDB),cooldownthebottletoroomtemperaturebeforeusing.

Wash Buffer (WDB)Providetheconcentratedsolution.Add 160 ml of >99%ethanol into thebottle andmixwith inversion thebottle gently at thebeginningofuse.AbottleofWDBisavailablefor12samplespreparation.

Requirements of Wash Buffer (WDB) with >99% ethanol and Elution Buffer (CDB)Prepare the requirementsofWash Buffer (WDB)with>99%ethanol andElutionBuffer (CDB)accordingtothenumberofsamplesforisolation;refertothefollowingtable.SetthebottleontheQuickGene-610L.(Seetheuser’sguideofQuickGene-610L.)PutappropriateamountofCDB into50mlcentrifuge tubeandset the tubes in theQuickGene-610Ltubeholder.(Seetheuser’sguideofQuickGene-610L.)

Table2BuffervolumeandthenumberofsamplestosetintheQuickGene-610L

Number of samples WDB with Ethanol CDB

6 160ml (1/2bottle) 11ml

12 320ml (1bottle) 16ml

18 480ml (11/2bottles) 24ml

24 640ml (2bottles) 32ml

30 800ml (21/2bottles) 40ml

36 960ml (3bottles) 48ml

42 1120ml (31/2bottles) 56ml

48 1280ml (4bottles) 64ml

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8-2 Sample preparations

•TheQuickGeneDNAwholebloodkitLisspecificallydesignedforgenomicDNAisolationfrom2mlofwholeblood.

•RecommendusingthewholebloodcollectedinEDTA·2Na,EDTA·2Korheparin.•Theyieldwilldependonthesamplecondition.•Usethekitatroomtemperature(15-30°C).Whenusingthekitat lowerorhighertemperatures,

theexpectedyieldmaynotbeobtained.•Accuratelymeasurethebuffervolumeduringtheexperiments.

<Preparationworkflowfromwholeblood>

Empty15ml(or50ml)Centrifugetube

Lysate

GenomicDNA

AddEDBsolution:300μl*1aAddwholeblood:2ml*1bAddLDB:2.5ml*1c

Mixthoroughlywithshaking10timesupanddown.Mixthoroughlybyvortexing(maximumspeedor>2,500rpm)for15sec.

Add>99%Ethanol:2.5ml

Mixthoroughlywithshaking10timesupanddown.Mixthoroughlybyvortexingmixer(maximumspeedor>2,500rpm)for15sec.

TransferthewholelysateintothecartridgeofQuickGene-610L

Select“DNAWHOLEBLOOD”modePress[START]Button

Defaultelutionvolume:500μl

1.

4.

2.

5.

6.

Incubatewithwaterbathat56°C,5min.3.

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Notice

1.Followtheprotocolof1ato1cexactly.Ifyouchangetheprotocol,maybereducedtheyield. Youcanuse50mlcentrifugetubeinsteadof15mltube.

1a.Add3.3mlofnuclease-freeultrapurewatertothevialcontainingthefreeze-driedprotease,anddissolveitcarefully.

Putthe300μlofEDBtobottomof15mltube.1b.Add2mlofwholebloodintothe15mltube,andthenadd2.5mlofLDBimmediately. (LeavingthesampleslongtimebeforeadditionofLDBmaybereducedtheyield.)1c.MixthesampleandLDBwithshaking10timesupanddown. ItisveryimportanttomixthoroughlythesampleafteradditionofLDB.

2.Vortexingfor15sec.withmaximumspeed. Recommendingvortexspeedis2,500rpmandmore. Incompletemixingatthistime,thesamplewillbecloggedthecartridgeofQuickGene-610L,or

lowyield.

3.Incubatewithwaterbathat56°C5min.Themaximumincubationtimeis10min. Whenyouusetheheatingblock,youhavetoincubateat56°C30min.

4.Add2.5ml>99%Ethanolandmixthesamplewithshaking10timesupanddown. Vortexingfor15sec.withmaximumspeed. Recommendingvortexspeedis2,500rpmandmore. Incompletemixingatthistime,thesamplewillbecloggedthecartridgeofQuickGene-610L,or

lowyield.

5.TransferthewholelysatetothecartridgeofQuickGene-610L.Performisolationwithin30min.afterlysatepreparation.

Ifaggregatesarepresentinthelysate,applythemalongwiththelysatetothecartridge.

6.Defaultelutionvolumeis500μl.Incaseofsettingtolessthan500μl,yieldmaydecline. ThestandardyieldofelutedgenomicDNAis30-80μgfrom2mlwholeblood. StoretheelutedgenomicDNAat-20°Cforlongstorage. Two timeselutionprogramcan increase the yield ofDNA for 10-20%withanother 500μl

ElutionBuffer(CDB)(totalElutionBuffer(CDB)volumeistobe1ml).Pleasereferto8-3andtheuser’sguideofQuickGene-610Lforsettingtheprogram.

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8-3 Genomic DNA isolation using the QuickGene-610L

Notice:Systemsetupandbasicoperations. Please read theuser’s guide of QuickGene-610L circumstantially for thedetails before

usingthesystem.

(1) Selection of isolation mode Select“DNAWHOLEBLOOD”modeforgenomicDNAisolationfromwholebloodwiththekit. (SeeAppendix1) Setting for the two times elution program:Change the parameter of “ELUTCOUNT” in the

“EXPERT”mode from“1” to “2”.Please refer to theuser’s guideofQuickGene-610L forchangingtheparameters.

Notice:Incorrectparametersin“EXPERT”modemaydamagetheinstrumentandwastesamples.

(2) Setting of cartridges and tubes Openthefrontcoverof the instrumentandset thecollectiontube(1.5mlmicrotube) in the

TubeHolderandWasteTube(WTL)intoHolderCarriage. •Usethe1.5mlmicrotubeforelutionandWasteTube(WTL)includingthekitforwaste. •UsethespecifiedCartridges(CAL2).

Notice:Refertotheuser’sguidefortheQuickGene-610Lfordetailsofsettingcartridges,tubesandbottles. Incorrectcartridgeplacementmayresultinthesolutionspillingorimproperisolation. Wearglovesduringtheexperimentstoavoidnucleasecontamination.

(3) Setting of reagents Prepare the required volume (see8-1Preparationof reagents) ofWashBuffer (WDB)with

>99%ethanol and ElutionBuffer (CDB) into the tubes; set them to the holder; and put theholdertothedesignatedpositionsofinstrument.

Notice:Wearglovesduringthehandlingofreagentstoavoidnucleasecontamination. •Refertheuser’sguidefortheQuickGene-610Lfordetailsforsettingreagents.

(4) Discharge Set the “DischargeTray” and check theTubeHolder andCartridgeHolder setting for the

correctpositions. Pressthe[DISCHARGE]afterclosedthefrontcoveroftheinstrument.

Notice:Becauseofairinthelines,incorrectvolumeofreagentsmayoccurwithoutdischargeoperation.

(5) Applying the prepared samples Apply all contents of prepared lysate samples (see 8-2 Sample preparations) into the each

Cartridge(CAL2)decantationorusingmicropipettes(anyaggregatesinthelysateshouldbetransferredintothecartridge).PleasenotethatdonotputlysateontheedgeofCartridge.

Put the capof theCartridgeHolder onto Cartridgeand rock itwith two ratchets.Set theCartridgeHolderontotheHolderCarriage.

(6) Isolation Check if the materials—Wash Buffer (WDB) with >99% ethanol, Elution Buffer (CDB),

Cartridges (CAL2) including samples,WasteTubes (WTL), and collection tubes are wellsetting.

Closethefrontcoveroftheinstrument. Confirmtheappropriatemodeontheoperationpanelandpressthe[START]button.

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(7) Collection of genomic DNA Aftercompletingtheprocess,eachsampleresultisindicatedontheoperationpanelasfollow; [v(Check)]:Completednormally [–(Hyphen)]:Notcompletednormally [_(Underscore)]:Nocartridgeornosample

Openthefrontcoverandremovethecollectiontube(s)fromtheTubeHolder. •Asgenomic DNA is eluted from the Cartridge(s) (CAL2) using 500μl ofElution Buffer

(CDB),thevolumeofrecoveredtotalDNAsolutionwillbe500μl. CoverwiththecapsonthecollectiontubecontainingtheisolatedgenomicDNA.

(8) Clean up Remove theWasteTubes (WTL) and dispose the waste fluid according to applicable

regulations. RemovetheCartridgeHolderanddisposetheCartridges(CAL2).

Warning: Disposalofwastefluidandconsumables. Whenusingthepotentiallyinfectioussamplesforexperiments,disposethemaccording

toapplicableregulations.

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9. TroubleshootingReviewtheinformationbelowtotroubleshoottheexperimentswithQuickGeneDNAwholebloodkitL.Forsystem-relatedproblems(e.g.,whenanerrormessageappears),seetheQuickGene-610Luser’sguide.

(1) Low yield or no DNA obtained

Cause Possible Solution

Reagents and whole bloodaddedinthewrongorder

Add the reagentsand samples to 15ml tube in the following orderwhen preparing the lysate:Protease (EDB: dissolved in 3.3 ml ofnuclease-freewater)➝wholeblood➝LysisBuffer(LDB).

I nsu f f i c i en t d i sso lu t i on o fprotease(EDB).

Addnuclease-freeultra pure water, and vortex thebottle. Leave thesolution30-40minutesandmix it a few times.Make sure if all thepowderinthesolutionisdissolvedcompletelybeforeuse.

Excess amount of samplewasused

Reducetheamountofwholebloodtobelowthespecifiedamount.

Excess amount of leukocytecells

A sample contained over 2×107 of leukocyte cells, the yield maydecrease. In thecaseofsample,dilute thesamplenotover2×107byPBS.

Excess amount of leukocytecells

A sample contained over 2×107 of leukocyte cells, the yield maydecrease. In thecaseofsample,dilute thesamplenotover2×107byPBS.

Insu f f i c i en t d i sso lu t i on o fprotease(EDB).

Addnuclease-freeultra pure water, and vortex thebottle. Leave thesolution30-40minutesandmix it a few times.Make sure if all thepowderinthesolutionisdissolvedcompletelybeforeuse.

InsufficientmixingattheadditionofLysisBuffer(LDB)

MixsampleimmediatelyafterLysisBuffer(LDB)addition,shakingtube10timesupanddownandvortexingfor15sec.withmaximumspeed.Recommendingvortexspeedis2,500rpmandmore.

Requirement volume of ethanolwas not added toWash Buffer(WDB)

Alwaysconfirm that the requiredvolumeofethanolwasadded to theWashBuffer(WDB)priortouse.

O l d W a s h B u f f e r ( W D B :includingethanol)used

Flash remainingWashBuffer (WDB: includingethanol)whichmaybeonedayoldormoreintheinstrumentpriortouse.StoretheWDBwithcapforlongstorage.

InsufficientmixingattheadditionofEthanol

Mixsample immediatelyafterEthanoladdition,shaking tube10timesup and down and vor texing for 15 sec. with maximum speed.Recommendingvortexspeedis2,500rpmandmore.

Lysate is not fully applied toCartridge(s)(CAL2)

Insufficientvortexing,aggregatesmaybepresentinthelysate.Mixthesamplethoroughly.

Insufficient amountsof reagentsused

Makesurethatsufficientamountofreagentareinthereagentbottles.

(2) Clogging the cartridge

Cause Possible Solution

Excess amount of samplewasused

Reducetheamountofwholebloodtobelowthespecifiedamount.

InsufficientmixingattheadditionofLysisBuffer(LDB)orEthanol

Mix sample immediately after Lysis Buffer (LDB) orEthanol addition,shaking tube 10 times upanddown and vortexing for 15 sec.withmaximumspeed.Recommendingvortexspeedis2,500rpmandmore.

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(3) Subsequent experiments (e.g., PCR) unsuccessful

Cause Possible Solution

Improperamount ofDNAusedforsubsequentexperiments

Determinetheconcentrationbasedontheabsorbanceat260nm.

(4) Supplying the precipitates in reagents

Cause Possible Solution

Storedatlowtemperature Storesolutionsat15-28°C.Iftheprecipitatesarecontained,incubatethebottleinawaterbathat37°C and mix with inversion the bottle intermittently until theprecipitatesaredissolved.

(5) The collection tubes are empty after the elution

Cause Possible Solution

Missedthedischarge Set the “DischargeTray” and check theTube Holder andCartridgeHoldersettingupintocorrectpositions.Pressthe[DISCHARGE]afterclosedthefrontcoveroftheinstrument.SeetheQuickGene-610Luser’sguide.

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10. Ordering InformationCat #Product

QuickGeneDNAwholebloodkitL DB-L

QuickGene-610LAutomaticNucleicAcidIsolationSystems

DedicatedreagentkitforQuickGene-610LtoisolatetheGenomicDNAfromwholeblood

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Appendix 1 “DNA WHOLE BLOOD” mode is set in the following parameter.

PARAMETER SET VALUE

DNA WHOLE BLOOD

BINDPEAK

WASHCOUNT

WASHPEAK

WASHVOL1

WASHVOL2

WASHVOL3

WASHVOL4

WASHVOL5

WAS2COUNT

WAS2PEAK

WAS2VOL1

WAS2VOL2

WAS2VOL3

WAS2VOL4

WAS2VOL5

ELUTVOL

ELUTPEAK

120

3

90

7500

6500

5500

0

0

0

90

7500

6500

5500

0

0

500

100

DB-L_HB-E_V20

Bio-Medical DepartmentKurabo Neyagawa Techno Center 3F, 14-5, Shimokida-Cho, Neyagawa, Osaka 572-0823, Japan TEL +81-72-820-3079 FAX +81-72-820-3095URL; http://www.kurabo.co.jp/bio/English/

✽Trademark and exclusion item Right to registered name etc. used in this handbook is protected by law especially even in the case of no denotation.