HiSpeed Plasmid Kits

超快速纯化至多750 µg转染级纯质粒或柯斯质粒DNA

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HiSpeed Plasmid Midi Kit (25)

Cat. No. / ID: 12643

25 HiSpeed Midi Tips, 25 QIAfilter Midi Cartridges, 25 QIAprecipitator Midi Modules plus Syringes, Reagents, Buffers.

试剂盒类型

Midi

Maxi

Inquire

HiSpeed Plasmid Kits 旨在用于分子生物学应用。这些产品不能用于疾病诊断、预防和治疗。

✓ 全天候自动处理在线订单

✓ 博学专业的产品和技术支持

✓ 快速可靠的（再）订购

特点

少于60分钟制备时间
无需离心即可澄清裂解液并进行异丙醇沉淀
在沉淀过程中不会损失DNA
可纯化至多750 µg高拷贝质粒DNA
使用LyseBlue，获得最佳的裂解效果和最大的DNA产量

产品详情

HiSpeed Plasmid Kits使用阴离子交换技术，超快速、大规模纯化得到质粒DNA，无需离心。得到的DNA相当于两次CsCl梯度离心处理得到的纯度，适合转染级纯度的应用。

绩效

HiSpeed Plasmid Kits提供QIAfilter Cartridges、HiSpeed Tips和QIAprecipitator模块，用于快速、大规模的制备质粒，无需真空底座。针筒规格的QIAfilter和QIAprecipitator模块取代了传统阴离子交换过程中的离心操作，质粒纯化更加方便、快捷。可从150–250 ml或50 µl – 150 ml的培养体积中纯化多至750 µg（Maxi）、或200 µg（Midi）的高拷贝质粒DNA（培养体积取决于质粒拷贝数、插入片段大小、宿主菌和培养基）。HiSpeed Tip支持高流速，确保更快地完成DNA结合、洗涤和洗脱。

此外，HiSpeed tips中新型的阴离子交换树脂广泛用于核酸的纯化。其独特的分离特性使得到的DNA的纯度相当于两次氯化铯梯度离心处理得到的纯度。

原理

QIAfilter Cartridges（参见"QIAfilter Cartridge"）是一种特制的过滤器，专用于取代细菌细胞碱裂解之后的离心步骤。QIAfilter Cartridges可去除SDS沉淀，只需很短的离心时间即可澄清细菌裂解液。预装的HiSpeed tips依靠重力流运行，确保不会流干，减少了质粒制备的手工操作时间。

独特的QIAprecipitator模块（参见"QIAprecipitator module"）取代了异丙醇沉淀后传统的离心法纯化DNA。异丙醇混合液流过QIAprecipitator的时候DNA会结合上去，然后用TE缓冲液或水将DNA从QIAprecipitator洗脱下来。这一独特的模块降低了离心后转移上清液过程丢失DNA的风险。

程序

经中和的细菌裂解液放入QIAfilter Cartridge中，几秒内过滤澄清。滤出液放至HiSpeed tip进行质粒DNA 纯化（参见"QIAGEN Plasmid Kit procedures"）。洗脱所得DNA与异丙醇混合并用注射器装入QIAprecipitator中。经浓缩和脱盐后，直接从QIAprecipitator上用TE缓冲液或水洗脱DNA。

应用

HiSpeed Plasmid Kits纯化得到超纯转染级纯DNA，适合于多种应用，包括：克隆、测序、转染和质粒介导的基因沉默等。

辅助数据和图表

QIAprecipitator module.

The unique QIAprecipitator module replaces the centrifugation step traditionally used to collect isopropanol-precipitated DNA following purification. The QIAprecipitator module traps the precipitated DNA while the isopropanol-buffer mixture flows through. The DNA is then simply eluted from the QIAprecipitator into a microcentrifuge tube with TE buffer or water. This unique module also eliminates the risk of pellet loss, which can occur during decanting of the supernatant following centrifugation.

资源

Download Safety Data Sheets for QIAGEN product components.

Publications

Characterization of Helicobacter pylori lytic transglycosylases Slt and MltD.

Chaput C; Labigne A; Boneca IG;

J Bacteriol; 2006; 189 (2):422-9 2006 Nov 3 PMID:17085576

Identification of a lipase-linked cell membrane receptor for pigment epithelium-derived factor.

Notari L; Baladron V; Aroca-Aguilar JD; Balko N; Heredia R; Meyer C; Notario PM; Saravanamuthu S; Nueda ML; Sanchez-Sanchez F; Escribano J; Laborda J; Becerra SP;

J Biol Chem; 2006; 281 (49):38022-37 2006 Oct 10 PMID:17032652

Role of parathyroid hormone in the downregulation of liver cytochrome P450 in chronic renal failure.

Michaud J; Naud J; Chouinard J; Désy F; Leblond FA; Desbiens K; Bonnardeaux A; Pichette V;

J Am Soc Nephrol; 2006; 17 (11):3041-8 2006 Oct 4 PMID:17021269

A rapid functional assay for the human trace amine-associated receptor 1 based on the mobilization of internal calcium.

Navarro HA; Gilmour BP; Lewin AH;

J Biomol Screen; 2006; 11 (6):688-93 2006 Jul 10 PMID:16831861

Engineering of a xylose metabolic pathway in Corynebacterium glutamicum.

Kawaguchi H; Vertès AA; Okino S; Inui M; Yukawa H;

Appl Environ Microbiol; 2006; 72 (5):3418-28 2006 May PMID:16672486

FAQ

The composition of Buffer STE is:

100 mM NaCl
10 mM Tris-Cl, pH 8.0
1 mM EDTA

Buffer STE is a DNA resuspension and storage buffer used in QIAGEN Plasmid Kits for plasmid purification and in some plasmid supplementary protocols. Details on buffer preparation and storage are presented in Appendix B of the QIAGEN Plasmid Purification Handbook.

FAQ ID -415

Buffer QF is the elution buffer used in QIAGEN Plasmid Kits for plasmid purification and in QIAGEN Blood & Cell culture kits.

The composition of Buffer QF is:

1.25 M NaCl
50 mM Tris-Cl, pH 8.5
15% isopropanol (v/v)

To make 1 liter of solution, dissolve 73.05 g NaCl and 6.06 g Tris base in 800 ml distilled water. Adjust the pH to 8.5 with HCl. Add 150 ml pure isopropanol. Adjust the volume to 1 liter with distilled water. Store at 15–25°C

FAQ ID -413

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

Yes, when eluting DNA from the QIAprecipitator Modules of the HiSpeed Plasmid Kits, water or buffers commonly used to dissolve DNA (e.g., Tris) may be employed.

Note: Store DNA at -20°C when eluted with water as DNA may degrade in the absence of buffering and chelating agents.

FAQ ID -306

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

Redissolve the DNA by warming the solution slightly, e.g. incubate it at 37°C, and allow more time for redissolving.

FAQ ID -572

No. Ethanol is not recommended when using the QIAprecipitator module of the HiSpeed Plasmid Kits for DNA precipitation. The finer precipitates formed with ethanol are likely to clog the QIAprecipitator.

FAQ ID -354

Use 500 µl instead of 1 ml of Buffer TE for elution of plasmid DNA from the QIAprecipitator of the HiSpeed Plasmid Kits. If low copy plasmids are used, the lysates of two QIAfilter Cartridges can be filtered into one equilibrated HiSpeed Tip. HiSpeed Maxi Kits will result in higher DNA concentration than HiSpeed Midi Kits, because the QIAprecipitators in both kits (Midi- and Maxi Module) use the same elution volume.

FAQ ID -1061

When using the QIAprecipitator module of the HiSpeed Plasmid Midi- or Maxi Kits, make sure to dry the membrane by pressing air through the QIAprecipitator at least twice. Dry the outlet nozzle of the QIAprecipitator with absorbent paper. This will prevent carry-over of alcohol into the eluate and enable optimal performance of the extracted DNA downstream.

Do not load eluate from from several columns on the QIAprecipitator, and be sure that the correct precipitator size is used for the corresponding HiSpeed Tip. Do not replace the isopropanol with ethanol for precipitation, since the use of ethanol will lead to a finer precipitate that can clog the module.

To prevent breakage and leakage of the module it is important to avoid excessive force, bending, or twisting while attaching the QIAprecipitator to the syringe. Do not stress the inlet by resting one edge of the QIAprecipitator on a hard surface (e.g., the edge of a sink) and depressing the syringe plunger. Always apply gentle, even, pressure perpendicularly to the QIAprecipitator.

FAQ ID -144

When using the silica-based QIAprep Spin Miniprep Kit, a protocol is contained in the QIAprep Miniprep Handbook, in Appendix C: Special Applications. The protocol is called: 'Purification of plasmid DNA prepared by other methods'.

For our anion-exchange based Plasmid Purification Kits, a protocol can be accessed online at our Plasmid Resource Center, and is called 'Re-Purification of Plasmid DNA Prepared by Methods other than QIAGEN Tips'.

FAQ ID -1031

The composition of Buffer TE is:

10 mM Tris·Cl, pH 8.0
1 mM EDTA

Buffer TE is a commonly used DNA resuspension and storage buffer. It is supplied in QIAGEN's Endofree Plasmid Kits, and used for plasmid DNA resuspension in combination with other QIAGEN Plasmid Kits. Details on buffer preparation and storage are presented in Appendix B of the QIAGEN Plasmid Purification Handbook.

FAQ ID -416

The lowest elution volume that should be used for both the QIAprecipitator Midi and the Maxi Module provided in the HiSpeed Plasmid Kits is 500 ul. The official elution volume for both modules is 1 ml. If a higher DNA concentration is desired and a reduction in yield of up to 10% is acceptable, the elution volume can be reduced. Elution volumes smaller than 500 ul will lead to incomplete wetting of the QIAprecipitator membrane and further reduced DNA yields.

FAQ ID -307

A precipitate forming upon adding LyseBlue reagent to Buffer P1 is a normal observation. This precipitate will completely dissolve after addition of Buffer P2. Please be sure to shake Buffer P1 vigorously before use to completely resuspend LyseBlue particles.

FAQ ID -1045

No, the QIAprecipitator Midi and Maxi modules of the HiSpeed Plasmid Midi- and Maxi Kits are not interchangeable. Each module has been designed for different capacities and recoveries.

FAQ ID -308

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

No. Although both Buffers N3 of the QIAprep Spin Miniprep and P3 of the QIAGEN Plasmid Kits perform the neutralization step in an alkaline lysis procedure, they are completely different. Buffer N3 contains a proprietary formula that sets up binding conditions for the QIAprep Miniprep column's silica-gel-membrane. Buffer P3 sets up binding conditions for QIAGEN anion-exchange columns. Our website explains QIAGEN's nucleic acid purification technologies in more detail.

FAQ ID -310

The composition of Buffer P3 is:

3.0 M potassium acetate, pH 5.5

Buffer P3 is the neutralization buffer used in QIAGEN's anion-exchange based Kits for plasmid preparation. Details of buffer preparation and storage are presented in Appendix B of the QIAGEN Plasmid Purification Handbook.

FAQ ID -418

Buffer QC is the wash buffer used in QIAGEN Plasmid Kits for plasmid purification and in QIAGEN Blood & Cell Culture kits.

The composition of Buffer QC is:

1.0 M NaCl
50 mM MOPS, pH 7.0
15% isopropanol (v/v)

To make 1 liter of solution, dissolve 58.44 g NaCl, 10.46 g MOPS (free acid) in 800 ml distilled water. Adjust the pH to 7.0 with NaOH. Add 150 ml pure isopropanol. Adjust the volume to 1 liter with distilled water. Store at 15–25°C.

FAQ ID -412

It is important to completely mix and lyse bacterial cells with plasmid Buffers P1 and P2. Incomplete mixing results in sticky or slimy areas of lysate, which will clog the filter matrix. It is recommended to completely resuspend cells in Buffer P1 and vigorously mix after addition of Buffer P2. Also mixing after Buffer P3 addition needs to be complete to allow fluffy precipitation of cell debris, which will float up. If the white debris does not float, dislodge it from the QIAfilter barrel wall (e.g. using a sterile pipette tip). Otherwise it will collect on the filter matrix and can lead to clogging. Use of LyseBlue reagent will help to achieve proper mixing results.

FAQ ID -1060

Buffer QBT is the equilibration buffer used in QIAGEN Plasmid Kits for plasmid purification and in QIAGEN Blood & Cell culture kits.

The composition of Buffer QBT is:

750mM NaCl • 50 mM MOPS, pH 7.0
15% isopropanol (v/v)
0.15 % Triton® X-100 (v/v)

To make 1 liter of solution, dissolve 43.83 g NaCl, 10.46 g MOPS (free acid) in 800 ml distilled water. Adjust the pH to 7.0 with NaOH. Add 150 ml pure isopropanol and 15 ml 10% Triton X-100 solution (v/v). Adjust the volume to 1 liter with distilled water. Store at 15–25°C

FAQ ID -411