QIAGEN Large-Construct Kit

For purification of up to 50 μg BAC, PAC, and P1 DNA or up to 200 μg cosmid DNA, free of genomic DNA

Features

  • Efficient removal of genomic DNA contamination
  • Pure, transfection-grade large-construct DNA
  • Easy and simple procedure
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QIAGEN Large-Construct Kit (10)

Cat. No. / ID: 12462

10 QIAGEN-tip 500, Reagents, Buffers, ATP-Dependent Exonuclease
€367.00
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The QIAGEN Large-Construct Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Product Details

The QIAGEN Large-Construct Kit provides gravity-flow, anion-exchange columns for purification of large-molecular-weight DNA. A unique integrated ATP-dependent exonuclease digestion step ensures selective removal of contaminating genomic DNA. The purified DNA is equivalent to that obtained by 2 x CsCl gradient centrifugation and is suitable for transfection.

Performance

DNA purified using the QIAGEN Large-Construct Kit is free of genomic DNA contamination (see figure " Efficient removal of genomic DNA"). This removal of genomic DNA ensures accurate and reliable DNA quantitation, which enables sensitive experiments to be carried out under defined and reproducible conditions.
See figures

Principle

DNA purification using the QIAGEN Large-Construct  Kit uses an optimized gravity-flow procedure which yields DNA of significantly greater purity than that obtained with other commonly used methods. A unique integrated treatment with ATP-Dependent Exonuclease enables efficient removal of genomic DNA.

The unique anion-exchange resin in QIAGEN-tips, included in the QIAGEN Large-Construct Kit, is developed exclusively for the purification of nucleic acids. Its exceptional separation properties result in DNA purity equivalent or superior to that obtained by two successive rounds of CsCl gradient centrifugation. Prepacked QIAGEN-tips (see figure " Anion-exchange tips") operate by gravity flow and never run dry, minimizing the hands-on time required for plasmid preparation. The entire QIAGEN plasmid purification system avoids the use of toxic substances such as phenol, chloroform, ethidium bromide, and CsCl, minimizing hazard both to the user and the environment.

See figures

Procedure

Following alkaline lysis of up to 500 ml culture (see flowchart " QIAGEN Plasmid Kit procedures"), a unique integrated digestion step with ATP-dependent exonuclease provided with the kit, ensures selective removal of contaminating genomic DNA, as well as nicked or damaged construct DNA. The sample is then loaded onto the anion-exchange tip, where plasmid DNA selectively binds under appropriate low-salt and pH conditions. RNA, proteins, metabolites, and other low-molecular-weight impurities are removed by a medium-salt wash. Genomic DNA-free, pure plasmid DNA is eluted in high-salt buffer. The DNA is concentrated and desalted by isopropanol precipitation and collected by centrifugation.

See figures

Applications

DNA purified using the QIAGEN Large-Construct Kit is suitable for any application, including:

  • Subcloning and preparation of shotgun libraries
  • Direct sequencing of large-construct DNA
  • Transfection

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

Quick-Start Protocols (2)
Safety Data Sheets (1)
Download Safety Data Sheets for QIAGEN product components.

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
Are there important considerations for plasma generation and urine handling?

It is strongly advised to follow the recommendations for preparing sample material provided in the corresponding Protocol Sheet to ensure reliable results.

Plasma: It is recommended to perform plasma separation immediately after blood collection when using EDTA or citrate as anticoagulant to prevent the release of genomic DNA into the plasma fraction.

Urine: Because circulating cell-free DNA in non-stabilized urine samples is rapidly degraded after sample collection due to high nuclease activity, eluates may contain no DNA or exhibit low DNA concentration. Therefore, it is recommended to stabilize urine samples. Even when using stabilized urine, it is recommended to perform a centrifugation step immediately after stabilization to prevent the release of genomic DNA from cells. Alternatively, non-stabilized urine samples can be processed immediately after collection and centrifugation using ATL-pretreatment and automated DNA extraction as described in the corresponding Protocol Sheet.

FAQ ID - 3699
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