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R.E.A.L. Prep 96 Plasmid Kit

碱裂解法从质粒、柯斯质粒、BAC、PAC和P1中快速获得少量测序级纯DNA

Features

  • 优化操作流程,适合用于纯化质粒、柯斯质粒、BAC、PAC和P1样本
  • 最佳的DNA产量

Product Details

R.E.A.L. Prep 96 Kits提供96孔板纯化,适用于QIAvac 96或者BioRobot 3000和BioRobot 8000工作站上,纯化获得测序级纯质粒DNA。

Performance

Rapid Extraction Alkaline Lysis系统通过使用多孔模式,以经济且高通量的方法,从质粒、粘粒、BAC、PAC和P1中纯化DNA,覆盖从细菌培养液到纯化的流程。R.E.A.L. Prep 96 Plasmid Kit能够重复性纯化质粒DNA(参见"Reproducible yields"),适合于常规的高通量应用,包括自动测序(参见"Reliable sequencing of shotgun clones")和筛选,如酶切和微阵列分析。

使用R.E.A.L. Prep 96的质粒、粘粒、BAC和菌株
质粒  粘粒(45-50 kb)  BACs(80-120 kb) 菌株
 endA- endA+
pUC18/19 Lorist pBeloBAC DH5α
HB101
pBluescript Lawrist    
XL1-Blue
NM554
pGEM pWE15   SOLR  
pTZ19R pOU61   DH10B  

Principle

R.E.A.L. Prep 96 Kit提供了适用于高通量的自动纯化方法。已成功运用在不同的高拷贝质粒、低拷贝质粒、BAC和E. coli菌株(参见"Plasmids, cosmids, BACs, and bacterial strains used with R.E.A.L. Prep 96")。

R.E.A.L. Prep 96操作流程基于改良的细菌细胞碱裂解,然后通过QIAfilter模块纯化澄清裂解产物,并且通过异丙醇沉淀进一步浓缩DNA。得到的DNA在少量的Tris缓冲液中重悬、待用。所有步骤不需要使用苯酚、氯仿、CsCl和溴化乙锭。

Procedure

R.E.A.L. Prep 96通过结合使用真空和离心,平行成倍处理96个样本(参见"R.E.A.L. Prep 96 procedure")。该程序建立在改良的碱裂解液上,然后使用特殊过滤器QIAfilter 96,进行高效过滤。通常情况下,从1.3 ml LB培养液中至多获得7 µg质粒DNA,或者在TB或者类似的超富集介质中至多获得20 µg质粒DNA。使用优化的BAC方案,可以从2.5 ml细菌培养液中至多隔离出800 ng大质粒DNA。

在多孔环境下生长的培养物通过使用改良的碱裂解方式裂解获得。选择性的加热步骤进一步使得蛋白质和碳水化合物变性和沉淀,然后使用QIAfilter 96孔板真空过滤去除。在过滤物中的DNA可以用异丙醇沉淀收集。

所有样品具有相同的寄主和载体结合时,该操作效果最佳。不同的寄主载体菌结合应该根据不同的介质选择和培养时间进行单独优化。

R.E.A.L. Prep 96 Plasmid Kit提供QIAfilter 96孔板、增长块和其他小规模制备96倍数的质粒或者粘粒DNA的组分。R.E.A.L. Prep 96 Plasmid Kit采用QIAvac 96。采用R.E.A.L. Prep 96操作流程纯化BAC样本需要使用48孔块,为BAC克隆提供优化的培养条件。48孔块不包括在R.E.A.L. Prep 96 Plasmid Kit内。平行处理能够在60–75分钟内纯化96个样品。

Applications

R.E.A.L. Prep 96 Kit可纯化得到即用型质粒DNA,产量和质量特别适合下列应用:

  • 测序 
  • 筛选

Supporting data and figures

Specifications

FeaturesSpecifications
ApplicationsAutomated sequencing, restriction digestion, microarray etc.
Plasmid typePlasmids, cosmids, BACs
Culture volume/starting material1.3 ml culture volume
Samples per run (throughput)1–4 x 96 samples per run
TechnologyQIAfilter technology
ProcessingManual (vacuum or centrifugation)
Yield<10 µg
Time per run or prep per run70 min/plate

Resources

Publications

Parallel human genome analysis: microarray-based expression monitoring of 1000 genes.
Schena M; Shalon D; Heller R; Chai A; Brown PO; Davis RW;
Proc Natl Acad Sci U S A; 1996; 93 (20):10614-9 1996 Oct 1 PMID:8855227

FAQ

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
Can Buffer R1 of the R.E.A.L. Prep 96 Plasmid Kit be replaced with Buffer P1?

Yes, it is possible to use Buffer P1 instead of Buffer R1 with the R.E.A.L. Prep 96 Plasmid Kit.

FAQ ID -823
Can BAC DNA prepared with the R.E.A.L. Prep 96 Plasmid Kit be used for high-throughput sequencing?

Yes. DNA prepared using the R.E.A.L. Prep 96 Plasmid Kit is well suited for high-throughput Capillary Electrophoresis (CE) DNA sequencing, or for fluorescent BAC DNA end sequencing on ABI PRISM 377 vertical slabgel sequencers. An optimized protocol for the purification of BAC DNA suitable for end sequencing on CE machines using R.E.A.L. Prep 96 is presented in QIAGENNews article 'High-throughput sequencing of plasmid and BAC DNA prepared using the REAL Prep 96 Plasmid Kit'. Typical sequence read lengths are around 600 bp.

High sample quality is essential for most sequencing applications on CE instruments. After intensive use of such sequencers (e.g., ABI PRISM 3700, MegaBACE 1000) in QIAGEN laboratories, a large set of statistical data on the quality and performance of DNA purified using the R.E.A.L. Prep 96 Plasmid Kit is available.

If you would like to take advantage of the Sequencing Service that we offer here at QIAGEN, please contact QIAGEN Technical Service.

FAQ ID -643
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