QIAprep 96 TuRSo Miniprep Kit

分子生物学実験グレードのプラスミドDNA(ウェルあたり最高20 μg)を精製

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QIAprep 96 Turbo Miniprep Kit (4)

Cat. No. / ID:  27191

For 4 x 96 high-purity plasmid minipreps, 4 each: TurboFilter 96 and QIAprep 96 Plates; S-Blocks, reagents, buffers, collection microtubes (1.2 ml), caps
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KitPlate
QIAprep 96 Turbo Kit
QIAprep 96 Turbo Core Kit
TurboFilter 96 Plates
QIAprep 96 Plates
For
QIAvac 96
BioRobot Universal
Preparations
4 x 96
24 x 96
QIAprep 96 TuRSo Miniprep Kitは分子生物学的アプリケーション用であり、疾病の診断、予防、あるいは治療に使用することはできません。

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Features

  • 能率的な精製
  • 70分以内で即使用可能なプラスミドDNA調製
  • 再現性のある分子生物学実験グレードのプラスミドDNA収量

Product Details

QIAprep 96 Turbo Kitsは、QIAprep 96とTurboFilter 96プレートを用いて分子生物学実験グレードのプラスミドDNA(ウェルあたり最高20 µg)の精製を実現します。

Performance

QIAprep 96 Turbo Kits は高純度なプラスミドDNAあるいはコスミドDNA(最高20 μg)が精製でき、精製されたDNAはシークエンシングおよびクローニングのようなルーチンの分子生物学アプリケーションに使用できます(図 "自動化シークエンシングに最適な高品質DNA")。

さらに、QIAprep 96 Turbo Kits は、大腸菌だけではなく、 ビール酵母菌、枯草菌、 Agrobacterium tumefaciens からのプラスミドDNA 精製にも利用することができます。これらのプロトコールに関しては弊社テクニカルサポートにお問い合わせください。

Principle

QIAprep 96ウェルプレート(図"QIAvac 96 ウェルプレート")は特殊なシリカメンブレンを使用しています。 高濃度のカオトロピック塩の存在下で20 μgまでのDNA はこのメンブレンに結合し、少量の低塩濃度のバッファーで溶出されます。QIAprepテクノロジーでは時間のかかるフェ ノール/クロロホルム抽出およびアルコール沈殿の必要がないだけではなく、樹脂漏れのリスクや、懸濁液取り扱いの難しさといったような問題もありません。QIAprep 96 プレートから溶出された高純度プラスミドDNA は、沈殿、濃縮、脱塩の必要がなく、即使用可能です。

TurboFilter 96プレートでは、高濃度のカオトロピック塩の存在下で溶解されたバクテリアライセートの迅速な清澄化用に特別にデザインされたフィルターユニットを使用しています。粗バクテリアライセートが遠心操作なしにQIAvac吸引装置を用いてTurboFilterでろ過されます。粒子を含まないろ過液は直接、精製、溶出用のQIAprepモジュールに注ぎ込まれます。TurboFilterテクノロジーによりQIAprepプラスミド精製が効率化されます。

Procedure

QIAprep 96 Turbo Miniprep Kits を用いたプラスミド精製法は、結合-洗浄-溶出という非常にシンプルなものです(フローチャート"QIAprep操作手順" 参照)。まずバクテリアを溶解した後、そのライセートをTurboFilter モジュールにより清澄化します。清澄化されたライセートをQIAprep 96 plateにアプライし、プラスミドDNAをシリカメンブレンに結合します。夾雑物を洗い流し、高純度DNAだけを少量の溶出バッファーまたは水で溶出します。

QIAprep 96 Turboの全操作を96ウェルフォーマットで行なうため、サンプル操作が最小限に抑えられ、96ミニプレップが45~70分で処理されます。キットに添付されている96ウェルブロックで、バクテリアを培養し、溶解することができます。QIAvac 96マニホールドは、96ウェルフォーマットでインラインろ過を行なうようにデザインされおり、清澄化されたライセートがTurboFilter 96プレートからQIAprep 96プレートのウェルへ直接流入して、96サンプルがハイスループットで同時に処理されます。

Applications

QIAprep 96 Turbo Miniprep Kit は、下記の用途を含むほとんどのアプリケーションに最適な高純度DNAを再現性良く調製します。

  • スクリーニング
  • PCR
  • シークエンシング
  • 制限酵素分解
  • ライゲーションやトランスフォーメーション

Resources

キットハンドブック (1)
Technical Information and Important Notes (1)
Supplementary Protocols (2)
This protocol is designed for 96 parallel plasmid DNA preparations from 1.3 ml overnight cultures.
MSDS (1)
Download Safety Data Sheets for QIAGEN product components.
Certificates of Analysis (1)
Kit Handbooks (1)

Publications

Systematic variation in mRNA 3'-processing signals during mouse spermatogenesis.
Liu D; Brockman JM; Dass B; Hutchins LN; Singh P; McCarrey JR; MacDonald CC; Graber JH;
Nucleic Acids Res; 2006; 35 (1):234-46 2006 Dec 8 PMID:17158511
Constraints on HIV-1 evolution and immunodominance revealed in monozygotic adult twins infected with the same virus.
Draenert R; Allen TM; Liu Y; Wrin T; Chappey C; Verrill CL; Sirera G; Eldridge RL; Lahaie MP; Ruiz L; Clotet B; Petropoulos CJ; Walker BD; Martinez-Picado J;
J Exp Med; 2006; 203 (3):529-39 2006 Mar 13 PMID:16533886
Regulation of gene expression in magnocellular neurons in rat supraoptic nucleus during sustained hypoosmolality.
Mutsuga N; Shahar T; Verbalis JG; Xiang CC; Brownstein MJ; Gainer H;
Endocrinology; 2004; 146 (3):1254-67 2004 Dec 9 PMID:15591143
Genome-scale cloning and expression of individual open reading frames using topoisomerase I-mediated ligation.
Heyman JA; Cornthwaite J; Foncerrada L; Gilmore JR; Gontang E; Hartman KJ; Hernandez CL; Hood R; Hull HM; Lee WY; Marcil R; Marsh EJ; Mudd KM; Patino MJ; Purcell TJ; Rowland JJ; Sindici ML; Hoeffler JP;
Genome Res; 1999; 9 (4):383-92 1999 Apr PMID:10207160
Positional cloning of the gene for X-linked retinitis pigmentosa 2.
Schwahn U; Lenzner S; Dong J; Feil S; Hinzmann B; van Duijnhoven G; Kirschner R; Hemberger M; Bergen AA; Rosenberg T; Pinckers AJ; Fundele R; Rosenthal A; Cremers FP; Ropers HH; Berger W;
Nat Genet; 1998; 19 (4):327-32 1998 Aug PMID:9697692

FAQ

What is the advantage of running an analytical gel with fractions of my plasmid preparation?

Running fractions saved from each step in the plasmid preparation procedure on an agarose gel enables monitoring the performance of each crucial step in the protocol. If the plasmid DNA is of low yield or quality, the samples can be analyzed to determine at what stage of the purification procedure the difficulty occurred.

Aliquots can be taken from the cleared lysate and the flow-throughs as indicated in the relevant protocols, precipitated with isopropanol and resuspended in a small volume of TE buffer.

Please see the Troubleshooting Section of the QIAprep Miniprep Handbook and Appendix A of the QIAGEN Plasmid Purification Handbook for instructions, and a picture and legend explaining the typical results you may see. You can also access this information on our Plasmid Resource Pages.

FAQ ID -769
How do I know if my plasmid is a high- or low copy number type?

Find out which origin of replication your plasmid contains, and look at the table below for classification into high-copy or low-copy types. This table can also be found online at the QIAGEN Plasmid Resource Center in the section 'Growth of bacterial cultures; Plasmid Copy Number' . A way to determine experimentally if the copy number of your plasmid is high or low is to perform a miniprep. A high-copy plasmid should yield between 3-5 ug DNA per 1 ml LB culture, while a low-copy plasmid will yield between 0.2-1 ug DNA per ml of LB culture.

 

Origins of replication and copy numbers of various plasmids and cosmids

DNA construct Origin of Replication Copy number Classification
Plasmids      
pUC vectors pMB1* 500–700 high copy
pBluescript® vectors ColE1 300–500 high copy
pGEM® vectors pMB1* 300–400 high copy
pTZ vectors pMB1* >1000 high copy
pBR322 and derivatives pMB1* 15–20 low copy
pACYC and derivatives p15A 10–12 low copy
pSC101 and derivatives pSC101 ~5 very low copy
Cosmids      
SuperCos pMB1* 10-20 low copy
pWE15 ColE1 10-20 low copy

* The pMB1 origin of replication is closely related to that of ColE1 and falls in the same incompatibility group. The high-copy plasmids listed here contain mutated versions of this origin.

FAQ ID -350
Can I eliminate RNase A from buffer P1 for my plasmid preparation to obtain RNase-free DNA for in-vitro transcription?
No, RNase A should not be omitted from buffer P1. It is required to prevent RNA contamination of the purified plasmid DNA. RNase A will not interfere with downstream in-vitro transcription experiments, since it will be efficiently removed during the plasmid purification procedures using QIAGEN Plasmid Kits.
FAQ ID -366
I left Buffer P1 at room temperature after addition of RNase A, what shall I do?

Buffer P1 with RNase A used in QIAGEN Plasmid Purification Kits should be fine at room temperature for a few days. We would expect the enzyme to have some residual activity. However, optimal results cannot be guaranteed after storage at room temperature. If you notice that RNase A activity is substantially reduced, you can add fresh RNase A to your buffer.

We recommend that Buffer P1 with RNase A be stored in the refrigerator (2–8°C). RNase A will be stable for 6 months under this condition.

FAQ ID -859
What is the recommended culture medium for the QIAprep System?
Luria-Bertani (LB) broth is the recommended culture medium for use with QIAprep Kits, since richer broths such as TB (Terrific Broth) or 2x YT lead to extremely high cell densities, which can overload the purification system. Please review the section 'Culture Media' of Appendix A in the QIAprep Miniprep Handbook or visit our Plasmid Resource Center for additional information on optimal plasmid culturing and extraction conditions for all QIAGEN Plasmid Purification Kits.
FAQ ID -154
What is the composition of Buffer P2?

The composition of Buffer P2 is:

  • 200 mM NaOH
  • 1% SDS (w/v)

It should be stored at room temperature. Buffer P2 is the lysis buffer used in a variety of QIAGEN kits for plasmid DNA purification. Details on buffer preparation and storage are presented in Appendix B of the QIAGEN Plasmid Purification Handbook.

FAQ ID -203
What is the volume of the eluate when using either spin or vacuum procedures with the QIAprep 96 Turbo Miniprep Kit?

The average eluate volume when using either spin or vacuum protocols with the QIAprep 96 Turbo Miniprep Kit is 60 µl. Since 100 µl Buffer EB (10 mM Tris-Cl, pH 8.5) or water are added to each well for elution, the dead volume per sample is approximately 40 µl for both vacuum and spin procedures.

FAQ ID -1067
Can I use QIAprep Miniprep kits for low-copy plasmids and cosmids?

All QIAprep Miniprep Kits can be used for preparation of low-copy number plasmids and cosmids up to 50 kb. Below are recommendations for processing low-copy constructs using QIAprep technology:

  • Use up to 10 ml overnight E. coli cultures grown in LB medium
  • Be sure to include the optional Buffer PB wash step for all bacterial strains
  • When plasmids or cosmids are larger than 10 kb, pre-heat Buffer EB (or water) to 70°C prior to eluting DNA from the QIAprep membrane
  • When using 10 ml culture volume, it is recommended to double the volumes of Buffers P1, P2, and N3

See also QIAGEN News 1998, Issue 5 for an article entitled 'Isolation of a low-copy plasmid from agrobacterium using QIAprep technology'. Alternatively, the R.E.A.L. Prep 96 Plasmid Kit can be used for high-throughput purification of larger plasmids (e.g., BACs, PACs, and P1s). See QIAGEN News 1999, Issue 2 for an article entitled 'High-throughput purification of BACs with the new R.E.A.L. Prep 96 protocol'.

FAQ ID -127
Can I use LyseBlue with R.E.A.L. Prep 96, QIAwell Ultra, or QIAprep 96 Turbo Miniprep Kits for the BioRobot systems?

For high-throughput BioRobot plasmid isolation systems such as R.E.A.L. Prep 96, QIAwell Ultra, or QIAprep 96 Turbo Miniprep, there is no need for visual lysis control with LyseBlue reagent, because

  1. insufficient resuspension and lysis have not been observed with these systems
  2. automation systems already handle the lysis steps well enough
  3. optical reading capabilities are not implemented on our BioRobot platforms
FAQ ID -863
What are the additional plasmid bands I see on my gel?

Open circular plasmid, resulting from single strand nicks, usually migrates slower in agarose gels and forms (faint) bands above the supercoiled plasmid DNA band. Sometimes an additional band of denatured supercoiled DNA migrates just below the supercoiled form. This form may result from prolonged alkaline lysis with Buffer P2 and is resistant to restriction digestion.

For a detailed description on how to run and interpret an analytical gel, please see Appendix A in the QIAGEN Plasmid Purification Handbook: "Agarose Gel Analysis of the Purification Procedure", or visit this link.

FAQ ID -1059
What is the composition of Buffer PB?
Buffer PB contains a high concentration of guanidine hydrochloride and isopropanol. The exact composition of Buffer PB is confidential. However, this buffer can be purchased separately: Buffer PB.
FAQ ID -2791
Why is my plasmid DNA yield low?

Low yields of plasmid DNA can be caused by a number of different factors. The most common causes for low yield are poor culturing conditions and plasmid propagation, excessive amounts of starting material resulting in insufficient bacterial cell lysis and column overloading. When working with the anion-exchange based QIAGEN Plasmid Purification Kits, extra care is required during the isopropanol precipitation step, as the glassy DNA pellet may be difficult to see, and tends to be only loosely attached to the side of the tube.

We strongly recommend to review the information provided on our Plasmid Resource Page in the section 'Optimal results with QIAGEN plasmid kits', as it provides useful background information on growing bacterial cultures and general considerations for optimal results. It is also necessary to follow the instructions in the relevant protocols precisely to ensure the best plasmid yield and quality.

To determine at what stage of the procedure any problem occurred, save fractions from different steps of the purification procedure, and analyze by agarose gel electrophoresis. For a detailed description on how to run and interpret an analytical gel, please see Appendix A in the QIAGEN Plasmid Purification Handbook: "Agarose Gel Analysis of the Purification Procedure", or visit the QIAGEN Plasmid Resource Center.

 

 

FAQ ID -768
Do you have a protocol for high-throughput purification of plasmid DNA using the BioRobot Universal System?
FAQ ID -1191
Do you have a protocol for isolation of plasmid DNA using the Sigma Centrifuge 4?

Yes, please follow the Supplementary Protocol 'Isolation of plasmid DNA (2x96) using the Sigma Centrifuge 4-15' (PR02).

-15
What is the RNase A concentration and composition of Buffer P1?

The composition of Buffer P1 is:

  • 50 mM Tris·Cl, pH 8.0
  • 10 mM EDTA
  • 100 µg/ml RNase A

After RNase A addition, the buffer should be stored at 2–8°C.

Buffer P1 is the resuspension buffer used in a variety of QIAGEN kits for plasmid DNA purification. Details on buffer preparation and storage are presented in Appendix B of the QIAGEN Plasmid Purification Handbook.

FAQ ID -198
What is the recipe for SOC medium?

The components of the SOC medium are:

  • 0.5% Yeast Extract
  • 2% Tryptone
  • 10 mM NaCl
  • 2.5 mM KCl
  • 10 mM MgCl2
  • 10 mM MgSO4
  • 20 mM Glucose*

*Note: add Glucose after autoclaving the solution with the remaining ingredients, and letting it cool down. Sterilize the final solution by passing it through a 0.2 µm filter.

SOC medium can be stored at room temperature and is stable for several years.

FAQ ID -798
What is the white insoluble precipitate in my resuspended plasmid DNA pellet?

White insoluble material in the resuspended plasmid DNA pellet indicates carry-over of salts and/or carbohydrates. Ensure that isopropanol is used at room temperature for precipitation. Some bacterial strains, such as TG1 and JM100, naturally produce a high level of carbohydrates. However, carbohydrate contamination may also be observed when using other strains. The most common cause of this problem is over-growth of bacterial cultures. To avoid this, closely follow the guidelines for Plasmid DNA Preparation in the Handbook that was provided with the respective QIAGEN Plasmid Kit.

Useful hints and information on optimizing plasmid preparations can be found at the QIAGEN Plasmid Resource Center.

FAQ ID -352
Why would clumps occur following the addition of Buffer P2 when using LyseBlue Reagent in a plasmid preparation?

Clumps that occur after addition of Buffer P2 in a bacterial lysate containing LyseBlue reagent indicate poor resuspension of the bacterial cell pellet in Buffer P1. This handling error leads to inefficient cell lysis, and incomplete precipitation of SDS, cell debris, and genomic DNA. When resuspending the cell pellet, vortexing longer or resuspending the pellet by pipetting up and down can help.

If cells have been resuspended properly in P1, “brownish areas” after P2 addition just indicate poor mixing of P1 and P2. To overcome this, continue mixing the solution by inverting it gently until a homogeneous blue suspension is achieved.

FAQ ID -862