QuickGene DNA whole blood kit L (DB-L) - wako-chem.co.jp · the Material Safety Data Sheet for...
Transcript of 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
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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.