QIAGEN Plasmid Kits
For purification of up to 10 mg transfection-grade plasmid or cosmid DNA
For purification of up to 10 mg transfection-grade plasmid or cosmid DNA
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Cat. No. / ID: 12123
✓ 24/7 automatic processing of online orders
✓ Knowledgeable and professional Product & Technical Support
✓ Fast and reliable (re)-ordering
QIAGEN Plasmid Kits provide gravity-flow, anion-exchange tips for purification of transfection-grade plasmid DNA. Lysate clearing and isopropanol precipitation are achieved by centrifugation.
The QIAGEN Plasmid Mega Kit (cat. no. 12181) and the QIAGEN Plasmid Giga Kit (cat. no. 12191) can be used with the QIAfilter Mega-Giga Cartridges (cat. no. 19781) as an optional protocol step for rapid clearing of bacterial lysates by filtration instead of centrifugation. See more information regarding principle below.
The QIAGEN Plasmid Kits uses gravity-flow QIAGEN anion-exchange tips for efficient purification of plasmid DNA. Up to
10 mg (giga), 2.5 mg (mega), 500 µg (maxi), 100 µg (midi), and 20 µg (mini) high-copy plasmid DNA is purified from culture (culture volumes depend on plasmid copy number, size of insert, host strain, and culture medium). Plasmid DNA purified with QIAGEN Plasmid Kits is highly suitable for use in applications such as transfection (see figure " Transfection efficiency versus plasmid purification method"), cloning, and in vitro transcription.
The unique anion-exchange resin in QIAGEN-tips 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 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.
As an optional protocol step for rapid clearing of bacterial lysates by filtration instead of centrifugation, you can use the QIAfilter Mega-Giga Cartridges (cat. no. 19781), which operate with house vacuum to efficiently clear even large volumes of bacterial lysate with minimal effort. The protocol is available in the handbook.
|Applications||Transfection, cloning, sequencing, capillary sequencing, etc.||Transfection, cloning, sequencing, capillary sequencing, etc.||Transfection, cloning, sequencing, capillary sequencing, etc.||Transfection, cloning, sequencing, capillary sequencing, etc.||Transfection, cloning, sequencing, capillary sequencing, etc.|
|Culture volume/starting material||3–10 ml culture volume||25–100 ml culture volume||100–500 ml culture volume||500 ml – 2.5 liters culture volume||2.5–5 liters culture volume|
|Plasmid type||High-copy, low-copy, cosmid DNA||High-copy, low-copy, cosmid DNA||High-copy, low-copy, cosmid DNA||High-copy, low-copy, cosmid DNA||High-copy, low-copy, cosmid DNA|
|Processing||Manual (gravity flow)||Manual (gravity flow)||Manual (gravity flow)||Manual (gravity flow)||Manual (gravity flow)|
|Sample per run||1 sample per run||1 sample per run||1 sample per run||1 sample per run||1 sample per run|
|Technology||Anion-exchange technology||Anion-exchange technology||Anion-exchange technology||Anion-exchange technology||Anion-exchange technology|
|Time per run||80 min||150 min||160 min||220 min||320 min|
|Yield||<20 µg||up to 100 µg||<500 µg||<2.5 mg||<10 mg|
With QIAGEN Plasmid Kits, bacterial lysates are cleared by centrifugation. The cleared lysate 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, and pure plasmid DNA is eluted in high-salt buffer (see flowchart " QIAGEN Plasmid Kit procedures"). The DNA is concentrated and desalted by isopropanol precipitation and collected by centrifugation.
Plasmid DNA purified with QIAGEN Plasmid Kits is highly suitable for use in applications, such as:
|Culture volume/starting material||3 ml–5 liters culture volume|
|Yield||<20 µg to <10 mg|
|Processing||Manual (gravity flow)|
|Samples per run (throughput)||1 sample per run|
|Time per run or prep per run||80–320 min|
|Applications||Transfection, cloning, sequencing, capillary sequencing etc.|
|Plasmid type||High-copy, low-copy, cosmid DNA|
The composition of Buffer STE is:
Buffer STE is a DNA resuspension and storage buffer used in QIAGEN Plasmid Kits for plasmid purification and in some plasmid supplementary protocols. Details on buffer preparation and storage are presented in Appendix B of the QIAGEN Plasmid Purification Handbook.
Yes, please follow the User-Developed Protocol 'Isolation of plasmid DNA from Citrobacter freundii using the QIAGEN Plasmid Midi Kit' (QP05).
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.
Yes, please follow the User-Developed Protocol 'Isolation of plasmid DNA from Lactobacillus spp. using the QIAGEN Plasmid Midi Kit' (QP07).
The composition of Buffer QF is:
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
LyseBlue reagent is provided in the following QIAGEN plasmid kits:
Yes, please follow the User-Developed Protocol 'Isolation of very low-copy plasmids from Streptomyces spp. using the QIAGEN Plasmid Maxi Kit' (QP13).
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.
Yes, please follow the User-Developed Protocol 'Isolation of plasmid DNA from Staphylococcus spp. using the QIAGEN Plasmid Midi Kit' (QP10).
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.
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.
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|
|500 ml||2.5 liters|
|Buffer P1||20 ml||125 ml|
|Buffer P2||20 ml||125 ml|
|Buffer P3||20 ml||125 ml|
|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.
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.
Yes, please follow either of the User-Developed Protocols:
Yes, please follow the Supplementary Protocol 'Isolation of endotoxin-free plasmid DNA using the QIAGEN Plasmid Midi Kit' (QP15).
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'.
The composition of Buffer TE is:
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.
Yes, please follow the User-Developed Protocol 'Isolation of plasmid DNA from Borrelia spp. using the QIAGEN Plasmid Midi Kit' (QP04).
Yes. Endotoxins can be removed from purified plasmid preparations by following the Supplementary Protocol 'Removal of endotoxins from purified plasmid DNA using the EndoFree Plasmid Maxi Kit' (QP12).
The plasmid DNA is first treated with endotoxin removal buffer ER, and then applied to QIAGEN's anion-exchange tip. After performing a wash step, the plasmid DNA is eluted from the tip, followed by isopropanol precipitation and redissolving the DNA in a suitable volume of endotoxin-free buffer TE.
Average endotoxin level in plasmid DNA purified with QIAGEN Plasmid kits, QIAfilter Plasmid kits and HiSpeed Plasmid purification kits is around 10 EU/µg DNA.
Yes, please follow the User-Developed Protocol:
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.
Yes, please follow the Supplementary Protocols:
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.
Yes, please follow the User-Developed Protocol 'Isolation of plasmid DNA from Proteus spp. using the QIAGEN Plasmid Midi Kit' (QP09).
Yes, please follow the User-Developed Protocol 'Isolation of Plasmid DNA from Corynebacterium glutamicum using the QIAGEN Plasmid Mini Kit' (QP06).
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.
Yes, please follow the User-Developed Protocol 'Isolation of plasmid from Oligotropha carboxidovorans using the QIAGEN Plasmid Midi Kit' (QP08).
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).
The composition of Buffer QC is:
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.
Yes, please follow the User-Developed Protocol 'Isolation of bacteriophage P1 derived constructs using the QIAGEN Plasmid Midi Kit' (QP03).
Yes, please follow the Supplementary Protocol 'Isolation of single-stranded DNA from M13 phage using QIAGEN Plasmid Kits' (QP14).
Maximum recommended culture volumes for standard Luria Bertani (LB) medium*:
(QIAGEN Plasmid Midi Kit)
(QIAGEN Plasmid Maxi Kit)
|25 ml||100 ml|
|100 ml||500 ml|
* For the QIAGEN-tip 100, the expected yields are 75–100 µg for high-copy plasmids and 20–100 µg for low-copy plasmids. For the QIAGEN-tip 500, the expected yields are 300–500 µg for high-copy plasmids and 100–500 µg for low-copy plasmids.
It is not recommended to use super rich growth media such as TB (terrific broth) or 2x YT for most commonly used high-copy plasmids. Although TB or 2x YT have the obvious advantage of producing more bacteria (2–5 times), this does not necessarily lead to greater yields or higher-quality DNA. Please visit the QIAGEN Plasmid Resource Center for further information on the growth of bacterial cultures.
The composition of Buffer QBT is:
To make 1 liter of solution, dissolve 43.83 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 and 15 ml 10% Triton X-100 solution (v/v). Adjust the volume to 1 liter with distilled water. Store at 15–25°C
When low-copy-number plasmids containing the pMB1 or ColE1 origin of replication are prepared using QIAGEN Plasmid Purification Kits, plasmid DNA yields can be improved by adding chloramphenicol to the culture medium (170 mg/liter) to amplify copy numbers. For details and precautions on the use of chloramphenicol when culturing plasmids, please refer to standard manuals for cloning procedures (e.g., Molecular cloning : A laboratory manual / T. Maniatis, E.F. Fritsch, J. Sambrook).
Cultures of bacteria containing low-copy-number plasmids amplified in the presence of chloramphenicol should be treated as if they contain high-copy-number plasmids when choosing the appropriate culture volumes for the QIAGEN-tip to be used.
Note that copy numbers of the current generation of plasmids are so high that selective amplification in the presence of chloramphenicol is not necessary to achieve high yields. Please see FAQ 350 for origins of replication and copy numbers of various plasmids.
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.