DOH-DL 15/16-021

313 Pages·2016·12.91 MB·English

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Neasy B!c?od & Tissue KH
DNeasy 96 BSood & Tissue Kit
For purincaiion ol tota! DNA from
animal biood
animai isssue
9. vl t
cultured cells
ixe< »
bacteria
QIAGEN
DOH-DL 15/16-02RT1I Document No. 1
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Trademarks: QiAGEN* DNeosy*, KNeasy* (QIAGEN Group); DU* (Beckman instrumenis. Inc.); hvacP (Matrix Technologies
Corp.); Triton® (Rohm tKfd Haas Company).
The PCR process is cowCTed by the foreign counterparts oi U.S. Pc^enfeNos, 4,683,202 cmd 4,683,195 owned by F. Hoffmann-
La Roche Ltd. QtAzot lysis Recent is a subject of US Pa^il No. 5,346,994 and for^gn equivaierds. -'RNA/atef®" is a ^aclemoTk
of AMBiON, Inc., Au^m, Texas and is covered by vunows U.S. and foreign patents.
8 2006 QIAGEN. a8 ligfits reserved.
DOH-DL 15/16-02RT1I Document No. 2
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5
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Product WaitiBiity and Sciiismeiiati ©stdffaniae
Taciinicai AM;sScnce
,<**^ _ _B*
iyh 8
9
Principle and procedure 9
Description of protocois 12
EquipsionS cetd Raagsats I® E® Sug^risd try Ussr 13
impoE'tan'? ^eSas S5
Sample colection and storage 15
Starting amounts of samples 15
Maximum amouro of starting material 15
Very smal sampie sizes 15
QuanNftcation of starting material 17
Preparation of Buffer AW 1 and Buffer AW2 18
Buffer AL 18
Proteinase K 19
Copurification of RNA 19
Centrifugation (DNeasy 96 procedures) 20
Elution of pure nucieic acids 21
Expected yields 22
Purification of high-molecular-weight DNA
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Bbarf ay Cs^s
isasy 96 ?n;(ceo!) 31
&gi5on Q& "EnSaE DMA {TOWI
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iToioaois
Preireo&nsn!" foi- Po.';aSa-!.:inb®ilei8(J 'fissua y.'i
Preis'esatmen!' f®j' Fo.'ffiiaiin-^sioti Tissii® (r:j.'^"
Qflam-Megafr.?® A4
Preh^oEmsnS' for Snsm-PcsiSv® SccSeric .^
"s'i-oublesliooiiag ©uide 4;7'
A^pen^'ix A: DateniiiaoSaoii c^ViAl, ?uriiy, and S.&igA; ®f 9i^A >*^.
Appsn&ci B: Cieenmg S-BSesSts 54,
Qi-dsring InScfmoiion .55
Q!AG?:M Disi'ribssters and imsa?'2"i's W1
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rt,-T°" .-^ . -i . . r.
5i\^ inJ^* 'l'" ^(.j^. iL^;ir'U^j'\?i~''?iir .J"^E<
&11ssusKEB-
Nurabsr sS p»®§is 50 250
DNeasy Mini Spin Columns (coloHess)
in 2 ml Colection Tubes 50 250
Colection Tubes j2 ml) 100 500
Buffer ATL 10ml 50ml
Buffer AL* 12ml 54ml
BuferAWI (concenh-ate)*' 19ml 95 ml
Buffer AVV2 (concentrate)" i 3 ml 66 mi
Buffer AE 22m! 2 x 60 ml
Protainase K ? 1.25 ml 6m!
1
Contains a chaoftopic salt. Not compaSbte with disinfed'ng mts asntalning bleach. See |xige 8 For
safety informction.
Buffer AW 1 and Buifer AW2 are 5uppSied as concentrates. ethanol (96-100%) according to the bottle
labei before use to obtain c woHclng solu^on.
Conhains sodium azide as a presercafive.
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DNeas"- 96 3icod & Tisus Ki? w
CtSiWag no. !1 592
N'cniber e& preps 4x96 12x%
DNeasy 96 Plates 4 12
S-Blocks* 2 2
Colection Microtubes, 1.2 ml (racked) 4x96 12x96
Collection Microtube Caps 2xp2©%%5x(»2Qx8}
Elution Microtubes RS (racked) and caps 4x96 12x96
AirPore Tape Sheets 25 3x25
Buffer AL* 86ml 247ml
Buffer ATI 80ml 3 x 80 ml
Buffer AV\/1 (concentratej" 98ml 3 x 98 ml
Buffer AW2 (concentrate)'5 68 mi 3 x 68 ml
Buffer AE 2x 110ml 500ml
Proteinase K 2 x 7 m! 5 x 7 ml
96-Weil-Plate Register 4 12
Handbook 1
Reusable; see Appendix B (page 54} for leaning instructions.
Contains a chootropic salt. Not compatible with disinfectanfs containing bleach. See page 8 for safety
information.
Buffer AW1 and Buffer AW2 are supplied as concenlralas. Add ethanol ;96-100%) according to the bottle
label before use to obtain a working sc^utkwi.
Contains sodium azide as a preservative.
.sitec^gi®
DNeasy spin columns, DNeasy 96 plates, and all buffers should be stored dry, at room
temperature (15-25°C) and are stable for 1 year under these conditions.
DNeasy Blood & Tissue Kits contain a ready-to-use proteinase K solution, which is
supplied in a specially formulated storage buffer. Proteinase K is stable for at least
1 year after delivery when shared at room temperature. For storage longer than one
year or if ambient temperatures often exceed 25°C, we suggest storing proteinase K at
2-8°C.
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DNeasy Blood & Tissue Kits and DNeasy 96 Blood & Tissue Kits are intended for
research use. No claim or representation is intended to provide information for the
diagnosis, prevention, or treatment of a disease.
All due care and attention should be exercised in the handling of the products. We
recommend all users of QIAGEN® products to adhere to the NIH guidelines that have
been developed for recombinant DNA experiments, or to other applicable guidelines.
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QIAGEN guarantees the performance of all products in the manner described in our
product literature. Vne purchaser inust determine the suitability of the product for its
particular use. Should any product fail to perform satisfactorily due to any reason other
than misuse, QSAGEN will replace it free of charge or refund the purchase price. We
reserve the right to change, alter, or modify ony product to enhance its performance
and design. If a QIAGEN product does not meet your expectations, simply ca!l your
local Technical Service Department or distributor. We will credit your account or
exchange the product - as you wish. Separate conditions apply to QIAGEN scientific
instruments, service products, and to products shipped on dry ice. Please inquire for
more information.
A copy of QIAGEN terms and conditions can be obtained on request, and is also
provided on the back of our invoices, if you have questions about product specifications
or performance, please caii QIAGEN Technical Services or your locai distributor (see
back coveri.
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At QIAGEN we pride ourselves on the quaisfy and availability of our technical support.
Our Technical Service Departments are staffed by experienced scientists with extensive
practical and theoretical expertise in molecular biology and the use of QIAGEN
products. If you have any questions or experience any difficuities regarding DNeasy
Blood & Tissue Kits or QIAGEN products in general, please do not hesitate to contact
us.
QIAGEN customers are a major source of informah'on regarding advanced or
specialized uses of our products. This information is helphil to other scientists as well as
to th® ressarchsrs at Q!AGEN. Wa therefcrs sncouragB you ?o contact us if you havs
any suggestions abou^ product performance or new appiications and techniques.
For technical assistance and more information please call one of the QIAGEN Technical
Service Departments or beat distributors {see back cover).
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In accordance with QSAGEN's !SO<:ertified Quality Management System, each lot of
DNeasy Blood & Tissue Kits and DNeasy 96 Biood & Tissue Kits is tested against
predetermined specifications to ensure consistent product quality.
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When working with chemicals, always wear a suitable lab coat, disposable gloves,
and protective goggles. For more informaKon, please consult the appropriate material
safety data sheets (MSDSsj. These are ayaitabie online in convenient and compact PDF
format at www.qiaaen.com/ts/msds.asp where you can find, view, and print the MSDS
for each QIAGEN kit and kit component.
. eesdic soiuHsmsMsetSy ic fhe s<amp
Buffer AL and Buffer AW1 contain guanidsne hydrochioride, which can form highly
reactive compounds when combined with bleach. If liquid containing this buffer is spilt,
clean with suitable laboratory detergentand water, if the spilt liquid contains potentially
infectious agents, clean the affected area firs? with laboratory detergent and water, and
then with 1 % (v/yj sodium hypochlorite.
The following risk and safety phrases appiy to components of DNeasy Blood & Tissue
Kits and DNeasy 96 Blood & Tissue Kits.
Buffer AL and Buifsr AW t (concewiiiate)
Contains guanidine hydrochloride: harmful, imtant. Risk and safety phrases:* R22-
36/38,513-26^6-46
ProS^nuss K
Contains proteinase K: sensitizer, irritant. Risk and safety phrases:* R36/37/38-
42/43, S23-24-26-36/37
24-Siour emergency informotkm
Emergency medical information in English, French, and German can be obtained
24 hours a day from:
Poison Information Center Mainz, Germany
Tel:[phone]
R22: Harmful if swallowed; R36/37/38: Inflating to eyes, respiratory system and skin; R36/38: irritating
to eyes and skin; R42/43: May cause sensitization by inhalation and skin contact; S13: Keep away from
food, drink, and animal feedingshjffc; S23: Do not breathe spray; S24: Avoid contact with skin; S26: In
case of contact with eyes, rinse immediofsly with plenty of water and seek medical advice; S36: Wear
suitable protective clolhing; S36/37: Wtear suitable proiech've clothing and gloves; S46: If swalbwed,
seek medical advice imniedkitely^ and shew ccmtainer or labd.
8 DNeosy Biood & Tissue Handbook 07/2006
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DNeasy Blood & Tissue Kits are designed for rapid purification of total DNA (e.g.,
genomic, mitochondrial, and pathogen] from a variety of sample sources including
fresh or frozen animal tissues and cells, blood, or bacteria. DNeasy purified DNA is
free of contaminants and enzyme inhibitors and is highly suited for PCR, Southern
blotting, RAPD, AFLP, and RFLP appfications.
Purification requires no phenoi or chioroform extroction or aicohol precipitation, and
involves minima! handling. This makes DNeasy Blood & Tissue Kits highly suited for
simultaneous processing of muitipfe somp!es. For higher-fhroughput applications, the
DNeasy 96 Blood & Tissue Kit enables simuitaneous processing of 96 or 192 samples.
The buffer system is optimized to aibw direct cei! lysis foilowed by seiectsv® binding of
DNA to the DNeasy membrane. After lysis, fhe DNecsy Biood & Tissue spin-column
procedure can be completed in as little as 20 minutes. Using the DNeasy 96 Blood &
Tissue Kit, 96 or 1 92 samples can be processed in just 1 hour after lysis.
Simple cenfrifugafion processing comp!ete!y removes conhaminants and enzyme
inhibitors such as proteins and divotent cations, and oilows simultaneous processing of
muliiple samples in poraile!. In addition, DNeasy Blood & Tissue procedures are
suitable for a wide rang® of sample sizes.
Purified DNA is eluted in low-sait buffer or water, ready for use in downstream
applications. DNeasy purifisd DNA {ypically has an Awo/AwQ ratio between 1.7 and
1.9, and is up to 50 kb in size, with fragments of 30 kb predonninating. The DNeasy
procedure also efficiently recovers DNA fragments as small as 1 00 bp.
^iTG^ffiipte ssi^e. precsi^yr®
DNeasy Blood & Tissue procedurss are simpEe (see ffowchartj. Samples are first lysed
using proteinase K.* Buffering conditions are adjusteci to provide opKmal DNA-
binding conditions and the lysate is loaded onto the DNeasy Mini spin column or the
DNeasy 96 plate. During centrifugation, DNA is selectively bound to the DNeasy
membrane as contaminants pcss through. Remaining contaminants and enzyme
inhibitors are removed in two efficient wash steps and DNA ss then eluted in water or
buffer, ready for use. DNeosy purified DNA has Assc/Azso ratios of 1.7-1.9, and
absorbance scans show a symmetric peak at 260 nm confirming high purity.
Lysis efficiency can be improved by cei! dtsrup^on using a rotor-stator homogenizer, such as the QIAGEN
TissueRuptor, or a bead mil!, such as Ae Q1AGEN Tissuelyser. A suppienientary protocol allowing the
simultaneous disruption of up to 48 tissue samples using the QIAGEN 'Pssuetyser is availabie {rom
QIAGEN Technicai Services.
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The DNeasy membrane combines the binding properties of a silica-based membrane
with siinple microspin technology or with the QIAGEN 96-Weii-Plate Cents-ifugation
System. DNA adsorbs to the DNeasy membrane in the presence of high concentrations
of chaotropic salt, which remove water from hydrated molecules in solution. Buffer
conditions in DNeasy Blood & Tissue procedures are designed to enable specific
adsorption of DNA to the silica membrane and optimal removal of contaminants one!
enzyme inhibitors.
OMeasy Wood & Tissue Handbook 07/2006
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