QIAprep Spin Miniprep Kit
For purification of up to 20 µg molecular biology grade plasmid DNA
For purification of up to 20 µg molecular biology grade plasmid DNA
Cat. No. / ID: 27104
The QIAprep Spin Miniprep Kit is designed to isolate up to 20 µg high-purity plasmid or cosmid DNA for routine molecular biology applications, including fluorescent and radioactive sequencing and cloning. Even higher yields (up to 30 µg) can be achieved using the High-Yield Supplementary Protocol. The QIAprep Spin Miniprep Kit can be automated on the QIAcube Connect.
We recommend using this product together with QIAvac 24 Plus for optimal results.
QIAprep Spin Miniprep standard protocols can also be executed using the TRACKMAN Connected system, paired with PIPETMAN M Connected pipettes, both from Gilson. The TRACKMAN Connected system guides researchers through the QIAprep Spin Miniprep protocols while automatically adjusting the Bluetooth-enabled PIPETMAN M Connected pipette settings. Each protocol step is recorded to accelerate reporting by generating a comprehensive run report. Download more information.
In partnership with My Green Lab, we've also assessed the environmental impact of the QIAprep Spin Miniprep Kit (250). My Green Lab ACT labels are designed to evaluate and score products on several sustainability criteria. These include:
• responsible chemical management
• sustainable content within products and packaging materials
• disposal of the packaging at the end of life
Products are scored from 1 to 10 except for energy and water consumption, which are scored as 1 point per kWh or gallon, respectively. A low score means a lower environmental impact (see figures "QIAprep Spin Miniprep Kit ACT environmental impact factor label US, EU and UK").
For an eco-friendlier alternative to this kit, see our QIAwave Plasmid Miniprep Kit.
The QIAprep Spin Miniprep Kit enables purification of up to 20 µg molecular biology grade plasmid DNA or cosmid DNA for use in routine molecular biology applications such as PCR, sequencing and cloning. Versatile QIAprep 2.0 Spin Columns can be used either in microcentrifuges, on vacuum manifolds, or in the QIAcube (see figures "QIAprep 2.0 Spin Column handling options A, B, and C"). The vacuum procedure provides simplified handling and faster sample processing. QIAprep 2.0 Spin Columns can be vacuum processed using the QIAvac 24 Plus or any other commercial manifold with luer connectors. The QIAprep Spin Miniprep Kit can now also be fully automated on the QIAcube Connect (see figure " QIAcube Connect").
|Purification module||QIAprep 2.0 Spin Columns|
|Preparation time||24 minipreps in 30 minutes|
|Equipment required||Microcentrifuge or vacuum manifold; fully automatable using the QIAcube|
|Capacity of column reservoir||800 µl|
|Minimum elution buffer volume||50 µl|
|Culture volume for high-copy plasmids||1–5 ml|
|Culture volume for low-copy plasmids/cosmids||1–10 ml|
Purified DNA can be used in restriction digestion (see figure " Complete digestion with various restriction enzymes").
QIAprep 2.0 Spin Columns 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. High-purity plasmid DNA eluted from QIAprep 2.0 Spin Columns is immediately ready to use – there is no need to precipitate, concentrate, or desalt.
To enable faster and more convenient sample processing and analysis, gel loading dye is provided in the kit. GelPilot Loading Dye contains 3 tracking dyes (xylene cyanol, bromophenol blue, and orange G) to facilitate the optimization of agarose gel run time and prevent smaller DNA fragments migrating too far (see figure " GelPilot Loading Dye").
The QIAprep procedure.
|Applications||Fluorescent and radioactive sequencing (including capillary sequencing), ligation, cloning, transformation, etc.|
|Processing||Manual (vacuum or centrifugation) or automated|
|Plasmid type||High-copy, low-copy, cosmid DNA|
|Culture volume/starting material||1–10 ml culture volume|
|Elution volume||50 µl (minimal)|
|Time per run or prep per run||<30 minutes|
|Samples per run (throughput)||1–24 samples per run|
|Number of preps per run||1–24 samples per run|
Here is the protocol for preparing buffer MP:
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.
LyseBlue reagent is provided in the following QIAGEN plasmid kits:
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.
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'.
No. The maximum culture volumes recommended for QIAGEN's plasmid preparation kits still apply, and should be strictly followed. LyseBlue reagent now allows the user to monitor potential problems (insufficient bacterial cell resuspension and lysis as a consequence of overloading) early in the plasmid preparation process.
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.
QIAprep spin miniprep kit is based on silica extraction chemistry. Average endotoxin levels that we have observed for Silica gel slurry is around 1200 EU/µg DNA.
Yes, please follow either of the User-Developed Protocols:
Unfortunately, we do not have any compatibility data for using potassium phosphate-based buffers instead of TE or water for the elution of plasmid DNA from the spin columns of the QIAprep Spin Miniprep Kit.
However, below is a reference where cDNA was eluted from QIAquick PCR Purification Kit columns with potassium phosphate buffer (4 mM, pH 8.5), after replacing the wash buffer (PE) with 5 mM potassium phosphate (pH 8.5) containing 80% ethanol:
Wang HY, Malek RL, Kwitek AE, Greene AS, Luu TV, Behbahani B, Frank B, Quackenbush J, Lee NH. "Assessing unmodified 70-mer oligonucleotide probe performance on glass-slide microarrays." Genome Biol. 2003, 4(1): R5. Epub 2003 Jan 6.
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.
Too vigorous mixing of the bacterial lysate causes genomic DNA to appear in the eluate. The lysate must be handled gently after addition of buffers P2 and P3 to prevent shearing of chromosomal DNA. The culture volume needs to be reduced if the lysate is too viscous for gentle mixing.
Use of LyseBlue Reagent enables visualization of efficient bacterial cell resuspension as a prerequisite for complete lysis, thereby helping to avoid overloading of the columns and additional difficulties related to highly viscous lysates.
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'.
Yes, please follow the User-Developed Protocol 'Isolation of plasmid DNA from Agrobacterium using the QIAprep Spin Miniprep Kit; spin procedure' (PR03s).
Yes, it is possible to isolate plasmid DNA from mammalian cells using the QIAprep Spin Miniprep kit . The article in QIAGEN News 1995 No. 2, page 11, Isolation of plasmid DNA from mammalian cells using QIAprep kit, describes a procedure that requires only 30 minutes compared to the time-consuming and labor-intensive Hirt method.
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.
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.
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.
The composition of Buffer EB is:
Buffer EB is the elution buffer used in the QIAquick PCR, Gel Extraction, Nucleotide Removal Kits, and MinElute Kits for DNA cleanup, and the QIAprep Miniprep Kits for small-scale plasmid purification. The purified DNA can also be eluted in TE (10 mM Tris-Cl, 1 mM EDTA, pH 8.0), but the EDTA may inhibit subsequent enzymatic reactions.
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.