QIAprep 96 Turbo Kits
For purification of up to 20 µg molecular biology grade plasmid DNA per well
- Streamlined purification
- Ready-to-use plasmid DNA within 70 minutes
- Reproducible yields of molecular biology grade plasmid DNA
For purification of up to 20 µg molecular biology grade plasmid DNA per well
Cat. No. / ID: 27191
The QIAprep 96 Turbo Kits provide QIAprep 96 and TurboFilter 96 plates for purification of up to 20 µg molecular biology grade plasmid DNA per well.
For optimal results it is recommended to use this product together with QIAvac 96.
The QIAprep 96 Turbo Kits are designed for isolation of up to 20 µg pure plasmid or cosmid DNA for use in routine molecular biology applications, such as sequencing and cloning (see figure "High-quality DNA for automated sequencing").
In addition to plasmid purification from Escherichia coli, QIAprep 96 Turbo Kits can be used to purify plasmid DNA from Saccharomyces cerevisiae, Bacillus subtilis, and Agrobacterium tumefaciens. Contact QIAGEN Technical Services or your local distributor for protocols for these applications.
QIAprep 96 plates (see figure "QIAvac 96 with 96-well plate") contain a unique silica membrane that binds up to 20 µg DNA in the presence of a high concentration of chaotropic salt, and allows elution in a small volume of low-salt buffer. QIAprep membrane technology eliminates time consuming phenol–chloroform extraction and alcohol precipitation, as well as the problems and inconvenience associated with loose resins and slurries. Pure plasmid DNA eluted from QIAprep 96 plates is immediately ready to use — there is no need to precipitate, concentrate, or desalt.
TurboFilter 96 plates are specially designed to allow fast clearing of bacterial lysates generated under high concentrations of chaotropic salts. Crude bacterial lysates are vacuum filtered through the TurboFilter 96 plate, eliminating the need for centrifugation. Particle-free lysates flow directly into the wells of the QIAprep 96 plate, ready for purification and elution. TurboFilter technology streamlines QIAprep plasmid purification and allows the procedure to be fully automated on the BioRobot 3000, the BioRobot 8000, or the BioRobot Universal System.
|Features||QIAprep 96 Turbo Miniprep Kit||QIAprep 96 Turbo BioRobot Kit|
|Applications||Fluorescent and radioactive sequencing, cloning, etc.||Fluorescent and radioactive sequencing, cloning, etc.|
|Culture volume/starting material||1–10 ml culture volume||1–10 ml culture volume|
|Plasmid type||High-copy, low-copy, cosmid DNA||High-copy, low-copy, cosmid DNA|
|Processing||Manual (vacuum)||Manual (vacuum)|
|Samples per run; throughtput||48–96 samples per run||48–96 samples per run|
|Technology||Silica technology||Silica technology|
|Time per run||<45 min/plate||<70 minutes|
|Yield||<20 µg||<20 µg|
Plasmid purification using QIAprep 96 Turbo Kits follow a simple bind–wash–elute procedure (see flowchart "The QIAprep procedure"). First, bacterial cultures are lysed and the lysates are cleared by filtration through TurboFilter modules. The cleared lysates are then applied to the QIAprep 96 plate where plasmid DNA adsorbs to the silica membrane. Impurities are washed away and pure DNA is eluted in a small volume of elution buffer or water.
The QIAprep 96 Turbo procedure is performed entirely in 96-well format, reducing sample handling to a minimum, allowing 96 parallel minipreps to be processed in 45–70 minutes. Bacterial cultures can be grown and lysed in the 96-well blocks provided with the kit. The QIAvac 96 manifold is designed for in-line filtration in 96-well format, so that cleared lysates from the TurboFilter 96 plate flow directly into the wells of the QIAprep 96 plate, providing high-throughput of 96 samples in parallel.The QIAprep 96 Turbo BioRobot Kit is a special kit format optimized on the BioRobot 8000 (no longer available) or the BioRobot Universal System. The kit provides all components required for automated preparation of 96 high-purity plasmid minipreps.
QIAprep 96 Turbo Kits provide reproducible yields of pure DNA suitable for use in many applications, including:
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.
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|
|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|
* 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.
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.
The composition of Buffer P2 is:
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.
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.
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:
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'.
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.
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.
Yes, please follow the Supplementary Protocol 'Isolation of plasmid DNA (2x96) using the Sigma Centrifuge 4-15' (PR02).
The composition of Buffer P1 is:
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.
The components of the SOC medium are:
*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.
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.
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.