QIAGEN Large-Construct Kit

ゲノムDNAフリーのBAC、PAC、P1(最高50 µg)、コスミドDNA(最高200 µg)の精製用

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QIAGEN Large-Construct Kit (10)

Cat. No. / ID:  12462

10 QIAGEN-tip 500, Reagents, Buffers, ATP-Dependent Exonuclease
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$583.00
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QIAGEN Large-Construct 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

  • 効率的なゲノムDNAコンタミの除去
  • トランスフェクショングレードの高純度ラージコンストラクトDNA
  • 迅速かつ簡単な調製法

Product Details

QIAGEN Large-Construct Kitはオープンカラム方式の陰イオン交換カラムで、高分子DNAの精製を行ないます。ユニークなATP依存性エキソヌクレアーゼ分解ステップが組み込まれているので、混入したゲノムDNAが選択的に除去されます。精製したDNAは、CsCl密度勾配遠心操作を2回行なって得られる精製グレードに匹敵し、トランスフェクション等のアプリケーションに最適です。

Performance

QIAGEN Large-Construct Kitを用いて精製したDNAは、ゲノムDNAコンタミがありません(図" ゲノムDNAの効率的な除去")。ゲノムDNAを除去すると正確で確実なDNAの定量が実現することにより、一定の再現性のある条件下で高感度の実験を行なうことができます。
See figures

Principle

QIAGEN Large-Construct Kitを利用したDNA精製では、至適化されたオープンカラム調製法を利用し、汎用されている方法に比較して、有意に純度の高いDNAを精製可能です。ユニークなATP依存性エキソヌクレアーゼ処理が組み込まれているので、ゲノムDNAが効率的に除去されます。

QIAGEN Large-Construct Kitに含まれる、QIAGEN-tipの非常にユニークな陰イオン交換樹脂は核酸精製のみを目的として開発されました。本製品の優れた核酸分離能力により、CsCl密度勾配遠心操作を2回連続で行なって得たDNAの純度に匹敵、あるいはそれ以上の純度のDNAが調製されます。充填済みQIAGEN-tip(図" 陰イオン交換チップ")はオープンカラム方式を採用しており、プラスミド調製中にカラムが乾燥することがなく、作業者がカラムに付き添う必要がないので、実際の操作時間は短くてすみます。全てのQIAGENプラスミド精製システムでは、ユーザーおよび環境への影響が最小限となるように、フェノール、クロロホルム、臭化エチジウム、CsCl等の有害な試薬を一切使用していません。

See figures

Procedure

500 mlまでの培養液のアルカリ溶解に続いて(フローチャート" QIAGEN Plasmid Kit操作手順")、本キットでは、ATP依存性エキソヌクレアーゼによるユニークな分解が組み込まれています。この製法により、切断、損傷したDNAおよび夾雑物としてのゲノムDNAの選択的な除去が確実になります。サンプルを陰イオン交換チップ上にロードすると、適切な低塩あるいはpH条件でプラスミドDNAが選択的に結合します。RNA、タンパク質、代謝物、その他の低分子量の不純物は中濃度の塩による洗浄で取り除かれます。ゲノムDNAフリーの高純度プラスミドDNAは高塩濃度バッファーで溶出されます。イソプロパノール沈殿によりDNAが濃縮および脱塩され、遠心操作により回収されます。

See figures

Applications

QIAGEN Large Construct Kitを用いて精製したDNAは以下のようなアプリケーションに最適です。

  • サブクローニングやショットガンライブラリーの調製
  • ラージコンストラクトDNAのダイレクトシークエンシング
  • トランスフェクション

Supporting data and figures

Specifications

FeaturesSpecifications
Plasmid typeBAC, PAC, P1, cosmid DNA
ApplicationsSubcloning, transfection, sequencing etc.
ProcessingManual (centrifugation)
Culture volume/starting material<500 ml culture volume
Samples per run (throughput)1 sample per run
TechnologyAnion-exchange technology
Time per run or prep per run280 min
Yield<150 ug

Resources

クイックスタートプロトコール (2)
キットハンドブック (1)
MSDS (1)
Download Safety Data Sheets for QIAGEN product components.
Safety Data Sheets (1)
Certificates of Analysis (1)
Quick-Start Protocols (2)

Publications

Functional characterization of Kaposi's sarcoma-associated herpesvirus ORF45 by bacterial artificial chromosome-based mutagenesis.
Zhu FX; Li X; Zhou F; Gao SJ; Yuan Y;
J Virol; 2006; 80 (24):12187-96 2006 Oct 11 PMID:17035322
Autographa californica multiple nucleopolyhedrovirus nucleocapsid assembly is interrupted upon deletion of the 38K gene.
Wu W; Lin T; Pan L; Yu M; Li Z; Pang Y; Yang K;
J Virol; 2006; 80 (23):11475-85 2006 Sep 20 PMID:16987976
Identification of in vivo-induced bacterial protein antigens during human infection with Salmonella enterica serovar Typhi.
Harris JB; Baresch-Bernal A; Rollins SM; Alam A; LaRocque RC; Bikowski M; Peppercorn AF; Handfield M; Hillman JD; Qadri F; Calderwood SB; Hohmann E; Breiman RF; Brooks WA; Ryan ET;
Infect Immun; 2006; 74 (9):5161-8 2006 Sep PMID:16926408
Molecular characterization of a diagnostic DNA marker for domesticated tetraploid wheat provides evidence for gene flow from wild tetraploid wheat to hexaploid wheat.
Dvorak J; Akhunov ED; Akhunov AR; Deal KR; Luo MC;
Mol Biol Evol; 2006; 23 (7):1386-96 2006 May 4 PMID:16675504

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
What is the composition of buffer QF?

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
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
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
Do you have a protocol for the isolation of BAC DNA using the QIAGEN Plasmid Midi Kit?

Yes, please follow the User-Developed Protocol 'Isolation of BAC DNA using the QIAGEN Plasmid Midi Kit' (QP01).  However, we recommend that the QIAGEN Large-Construct Kit be used for the purification of BAC DNA as it contains an exonuclease buffer for the removal of gDNA.

FAQ ID -881
Do you sell the ATP-Dependent Exonuclease of the QIAGEN Large-Construct Kit separately?
Sorry, but we only sell the ATP-Dependent Exonuclease as a part of the QIAGEN Large-Construct Kit.
FAQ ID -825
What are the recommended culture and buffer volumes for a very low-copy plasmid?

Very low-copy plasmids and cosmids of less than 10 copies per cell often require large culture volumes to yield significant amounts of DNA. The recommended conditions below are suitable for QIAGEN-tip 100 or QIAGEN-tip 500, and use centrifugation to clear lysates rather than QIAfilter Cartridges, due to the large culture volumes. After alkaline lysis, there is an additional isopropanol precipitation step to decrease the amount of lysate before DNA is bound to the QIAGEN-tip. Please follow the protocol for 'Very Low-Copy Plasmid/Cosmid Purification Using QIAGEN-tip 100 or QIAGEN-tip 500' in the QIAGEN Plasmid Purification Handbook. Culture volumes and tip sizes are selected to match the quantity of expected DNA with the capacity of the QIAGEN-tip.

 

Parameters for purification of very low-copy plasmids and cosmids of less than 10 copies per cell

 

Required DNA yield* Up to 100 ug Up to 500 ug

Culture volume

500 ml 2.5 liters
Buffer P1 20 ml 125 ml
Buffer P2 20 ml 125 ml
Buffer P3 20 ml 125 ml

QIAGEN-tip

QIAGEN-tip 100

QIAGEN-tip 500

Buffer QBT (for equilibrating) 4 ml 10 ml
Buffer QC (for washing) 2x 10 ml 2x 30 ml
Buffer QF (for elution) 5 ml 15 ml

* For very low-copy plasmids, expected yields are 20–100 µg for the QIAGEN-tip 100, and 100–500 µg for the QIAGEN-tip 500.

† Volumes of lysis Buffers P1, P2, and P3 are higher than in the standard protocols in order to efficiently lyse the large number of cells required for purification of very low-copy plasmids and cosmids.

FAQ ID -168
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
What is the composition of buffer TE?

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
What to do if cell clumps are present after Buffer P2 addition when using LyseBlue Reagent?

If cell clumps are present after adding Buffer P2 to your samples when using a QIAGEN Plasmid Purification Kit in combination with LyseBlue Reagent, you have two options:

  • pipet the cell clumps up and down for resuspension
  • transfer any clumps to a separate tube, add Buffer P1 and mix vigorously for resuspension, add Buffer P2 for lysis, and subsequently transfer the lysed material back to combine it with the rest of the original solution

Note: Avoid incubation times longer than 5 minutes in Buffer P2 as this will irreversibly denature plasmid DNA. In the scenario above, Buffer P3 may need to be added to portions of the sample, which can be subsequently combined once resuspension, lysis and neutralization of all fractions is complete.

FAQ ID -861
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 composition of buffer P3?

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
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
Is plasmid DNA purified with QIAGEN Plasmid Purification Kits suitable for in vitro transcription?

Plasmid preparations are free of any detectable proteins or other contaminants when purified using QIAGEN's anion-exchange kits according to the recommended protocols. DNA purified using QIAGEN Plasmid Kits, QIAfilter Plasmid Kits, or EndoFree Plasmid Kits gives excellent results with in-vitro transcription experiments.

Although a high level of RNase A is employed at the beginning of the procedure, it is removed efficiently by potassium dodecyl sulfate precipitation and subsequent washing with Buffer QC. It is possible, although not necessary, to omit RNase A from the procedure when purifying DNA for in vitro transcription. In this case, increasing the volume of Wash Buffer QC is recommended (e.g., for a Midi preparation on a QIAGEN-tip 100, use at least 2x 30 ml of Buffer QC instead of 2x 10 ml).

FAQ ID -1
What is the composition of buffer QC?

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
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