QIAGEN Plasmid Kits

For purification of up to 10 mg transfection-grade plasmid or cosmid DNA

Features

  • Purity equivalent to 2 x CsCl gradient centrifugation
  • High yields of plasmid DNA
  • Cost-effective preparations
  • LyseBlue for optimum lysis and maximum DNA yield
QIAGEN Plasmid Mini Kit (25)

Cat. No. / ID: 12123

25 QIAGEN-tip 20, Reagents, Buffers
€171.00
KitBufferColumnCartridge
Plasmid Kit
Plasmid Buffer Set
QIAGEN-tip
QIAfilter Cartridge
Column type
Mini
Midi
Maxi
Mega
Giga
Preparations
25
100
Add to cart
QIAGEN Plasmid Kits are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

Product Details

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.

Performance

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.

See figures

Principle

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.

Specifications

Features
Plasmid
Giga Kit
Plasmid
Mega Kit
Plasmid
Maxi Kit
Plasmid
Midi Kit
Plasmid
Mini Kit
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 2.5–5 liters culture volume 500 ml – 2.5 liters culture volume 100–500 ml culture volume 25–100 ml culture volume 3–10 ml culture volume
Elution volume Variable Variable Variable Variable Variable
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 320 min 220 min  160 min 150 min 80 min
Yield <10 mg <2.5 mg <500 µg up to 100 µg <20 µg
 

 

Procedure

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. 

See figures

Applications

Plasmid DNA purified with QIAGEN Plasmid Kits is highly suitable for use in applications, such as:

  • Transfection
  • Cloning
  • PCR
  • In vitro transcription 

Supporting data and figures

Specifications

FeaturesSpecifications
culturevolumestartingmaterial3 ml–5 liters culture volume
technologyAnion-exchange technology
yield<20 µg to <10 mg
processingManual (gravity flow)
samplesperrunthroughput1 sample per run
timeperrunorprepperrun80–320 min
plasmidtypeHigh-copy, low-copy, cosmid DNA
applicationsTransfection, cloning, sequencing, capillary sequencing etc.

Resources

Supplementary Protocols (8)
This protocol is designed for the rapid, easy, and non-toxic preparation of up to 2 mg genomic DNA from not more than 2 g of tissue using QIAGEN-tip 2500. QIAGEN® Genomic-tips 20/G, 100/G, and 500/G can also be used with this protocol by reducing the amount of starting material according to the table on page 2. The purified genomic DNA ranges in size from 50-150 kb.
This protocol is designed for isolation of up to 200 μg RNA from 150 mg plant tissue or up to 1 mg RNA from 600 mg plant tissue and is for use with QIAGEN-tip 100 or QIAGEN-tip 500, respectively.
This protocol is for purification of up to 100 µg endotoxin-free plasmid DNA using QIAGEN-tip 100.
This protocol is designed to provide up to 150 μg BAC/PAC/P1 DNA or up to 400 μg cosmid DNA.
Endotoxin-free DNA is essential for gene therapy research and will improve transfection into sensitive eukaryotic cells.
User-Developed Protocols (12)
The procedure has been used successfully for isolation of different medium-copy-number plasmids carrying pHM1519 or pBL1 origins of replication from Corynebacterium glutamicum ATCC 13032. Yield of plasmid DNA was typically 0.4-1.5 µg per ml LB culture, although yield was dependent on the vector, the insert, and the size of the plasmid.
The procedure has been used successfully for isolation of high- and low-copy-number plasmids from various Bacillus subtilis strains. Yield of plasmid DNA was typically 10-20 µg plasmid DNA from 100 ml culture.
The procedure has been used successfully for isolation of 110 kb P1 DNA (pAdsacBII with an 80 kb insert) from Escherichia coli strain NS3529. Yield of P1 DNA was typically 10-50 µg from 500 ml culture.
The procedure has been used successfully for isolation of a variety of medium-copy-number shuttle vectors from S. xylosus, S. carnosus, S. epidermidis, and S. aureus. Yield of plasmid DNA was typically 2-10 µg from 50 ml culture.
The procedure has been used successfully for isolation of cryptic plasmids (pLC2-based) from mesophilic Lactobacillus strains such as L. sake and L. curvatus. Yield of plasmid DNA was typically 10-20 µg plasmid DNA from 100 ml culture.
This procedure has been used successfully for isolation of 150-250 kb BAC DNA from a mouse-BAC library cloned in pBeloBAC11 from Escherichia coli strain HB101/r. The yield of BAC DNA from 100 ml culture was typically 20-40 μg.
The procedure has been used successfully for isolation of high-copy-number plasmids from Proteus vulgaris and Proteus mirabilis. Yield of plasmid DNA was typically 3-8 µg DNA per ml culture.
The procedure has been used successfully for isolation of plasmids SCP2 and SCP2* as well as the plasmids listed in Table 1 (see next page) from S. coelciolor and S. lividans strains.
The procedure has been used successfully for isolation of the large (128 kb), very-low-copynumber (1-2 copies per cell) plasmid pHCG3 and its derivatives from Oligotropha carboxidovorans. Yield of plasmid DNA was typically 3-6 µg plasmid DNA from 200 ml culture.
The procedure has been used successfully for isolation of linear plasmids from Borrelia burgdorferi sensu lato species, which include Borrelia burgdorferi sensu stricto, Borrelia afzelli, and Borrelia garinii.
The procedure has been used successfully for isolation of high-copy-number plasmids from Citrobacter freundii. Yield of plasmid DNA was typically 3-8 µg DNA per ml culture.
Quick-Start Protocols (2)

Publications

Oncocytic change in pleomorphic adenoma: molecular evidence in support of an origin in neoplastic cells.
Di Palma S; Lambros MB; Savage K; Jones C; Mackay A; Dexter T; Iravani M; Fenwick K; Ashworth A; Reis-Filho JS;
J Clin Pathol; 2006; 60 (5):492-9 2006 Feb 7 PMID:16467165
Haploinsufficiency of C2GnT-I glycosyltransferase renders T lymphoma cells resistant to cell death.
Cabrera PV; Amano M; Mitoma J; Chan J; Said J; Fukuda M; Baum LG;
Blood; 2006; 108 (7):2399-406 2006 Jun 15 PMID:16778138
RepAM of the Amycolatopsis methanolica integrative element pMEA300 belongs to a novel class of replication initiator proteins.
Te Poele EM; Kloosterman H; Hessels GI; Bolhuis H; Dijkhuizen L;
Microbiology (Reading); 2006; 152 (Pt 10):2943-2950 2006 Oct PMID:17005975
Involvement of Bcl-X(L) deamidation in E1A-mediated cisplatin sensitization of ovarian cancer cells.
Chang CY; Lin YM; Lee WP; Hsu HH; Chen EI;
Oncogene; 2006; 25 (18):2656-65 2006 Apr 27 PMID:16331250

FAQ

What is the composition of buffer STE?

The composition of Buffer STE is:

  • 100 mM NaCl
  • 10 mM Tris-Cl, pH 8.0
  • 1 mM EDTA

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.

FAQ ID -415
Do you have a protocol for the isolation of plasmid DNA from Citrobacter freundii?

Yes, please follow the User-Developed Protocol 'Isolation of plasmid DNA from Citrobacter freundii using the QIAGEN Plasmid Midi Kit' (QP05).

FAQ ID -885
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
Do you have a protocol for the isolation of total RNA from plant tissue using QIAGEN-tips?
Yes, please follow the Supplementary Protocol 'Isolation of total RNA from plant tissue using the QIAGEN-tip' (QR01).
FAQ ID -949
Do you have a protocol for the isolation of plasmid DNA from Lactobacillus spp.?

Yes, please follow the User-Developed Protocol 'Isolation of plasmid DNA from Lactobacillus spp. using the QIAGEN Plasmid Midi Kit' (QP07).

FAQ ID -887
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
Which QIAGEN plasmid preparation kits will contain LyseBlue Reagent?

LyseBlue reagent is provided in the following QIAGEN plasmid kits:

FAQ ID -865
What is the recipe for LB?
Preparation of LB medium: Dissolve 10 g tryptone, 5 g yeast extract, and 10 g NaCl in 800 ml dH2O. Adjust the pH to 7.0 with 1 N NaOH. Adjust the volume to 1 liter with dH2O. Sterilize by autoclaving.
FAQ ID -212
Do you have a protocol for the isolation of very low-copy plasmids from Streptomyces spp.?
FAQ ID -893
How do I know if my plasmid is a high- or low copy number type?

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
Plasmids      
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
Cosmids      
SuperCos pMB1* 10-20 low copy
pWE15 ColE1 10-20 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.

FAQ ID -350
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
I left Buffer P1 at room temperature after addition of RNase A, what shall I do?

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.

FAQ ID -859
Do you have a protocol for the isolation of plasmid DNA from Staphylococcus spp.?

Yes, please follow the User-Developed Protocol 'Isolation of plasmid DNA from Staphylococcus spp. using the QIAGEN Plasmid Midi Kit' (QP10).

FAQ ID -890
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
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
How do I perform a DNA precipitation to concentrate my sample?
  • Add 1/10 volume of 3 M Na-Acetate pH 5.2, and 2 to 2.5 volumes of ice-cold 100% ethanol to the DNA sample
  • Mix, and store at -20°C for at least 1 hour to precipitate the DNA
  • Recover the precipitated DNA by centrifugation at full speed in a microcentrifuge for 15-20 minutes
  • Pour off the ethanol and wash the pellet twice with room-temperature 70% ethanol
  • Allow the DNA pellet to air-dry
  • resuspend the DNA in a suitable volume of sterile TE buffer or distilled water
FAQ ID -305
With LyseBlue reagent for lysis control, can I now process more bacterial culture and overload the columns?

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.

FAQ ID -864
What is the recipe for 2x YT?
2x YT medium, per liter 16 g tryptone 10 g yeast extract 5 g NaCl Media Preparation and Bacteriological Tools. Edited by: Fred M. Ausubel, Roger Brent, Robert E. Kingston, David D. Moore, J.G. Seidman, John A. Smith, Kevin Struhl Current Protocols in Molecular Biology (1994), Section 1.1.3.
FAQ ID -213
What are the additional plasmid bands I see on my gel?

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.

FAQ ID -1059
Do you have a protocol for the isolation of plasmid DNA from Bacillus subtilis?
How can I increase low-copy plasmid DNA yields using QIAfilter Plasmid Kits?

If low-copy plasmids are used with QIAfilter Plasmid Kits, the cell lysates of two QIAfilter Cartridges can be filtered into one previously equilibrated QIAGEN-tip, resulting in doubled DNA yields.

Similarly, if low copy plasmids are used with HiSpeed Plasmid Kits, the lysates of two QIAfilter Cartridges can be filtered into one equilibrated HiSpeed Tip.

FAQ ID -1062
895 - Do you have a protocol for the isolation of endotoxin-free plasmid DNA using the QIAGEN Plasmid Midi Kit?

Yes, please follow the Supplementary Protocol 'Isolation of endotoxin-free plasmid DNA using the QIAGEN Plasmid Midi Kit' (QP15).

What can I do when the DNA pellet prepared with QIAGEN Plasmid Kits has been overdried?
Redissolve the DNA by warming the solution slightly, e.g. incubate it at 37°C, and allow more time for redissolving.
FAQ ID -572
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
Why do I get genomic DNA contamination in my plasmid prep?

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.

Additional information for successful plasmid preparations using QIAGEN's broad selection of Plasmid Kits can be found at our Plasmid Resource Center.

FAQ ID -353
Where can I find a protocol for cleanup of already purified plasmid DNA?

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'.

 

FAQ ID -1031
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
Do you have a protocol for the isolation of plasmid DNA from Borrelia spp.?

Yes, please follow the User-Developed Protocol 'Isolation of plasmid DNA from Borrelia spp. using the QIAGEN Plasmid Midi Kit' (QP04).

FAQ ID -884
Do you have a protocol for the removal of endotoxins from already purified plasmid DNA?

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).

This protocol requires the EndoFree Plasmid Buffer Set, which can be purchased separately. Alternatively, the EndoFree Plasmid Maxi Kit, containing all necessary components, can be used.

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.

FAQ ID -500
What is the expected level of endotoxins in plasmid DNA purified with QIAGEN Plasmid kits, QIAfilter Plasmid kits and HiSpeed Plasmid purification kits?

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.

FAQ ID -3082
Do you have a protocol for the isolation of plasmid DNA from yeast?

Yes, please follow the User-Developed Protocol:

FAQ ID -853
I am seeing a precipitate after adding LyseBlue reagent to Buffer P1. What should I do about that?

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.

FAQ ID -1045
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
Do you have protocols for the isolation of genomic DNA from tissue using QIAGEN-tips 2500 (Mega) and 10000 (Giga)?
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
Can Buffers N3 and P3 be used interchangeably?
No. Although both Buffers N3 of the QIAprep Spin Miniprep and P3 of the QIAGEN Plasmid Kits perform the neutralization step in an alkaline lysis procedure, they are completely different. Buffer N3 contains a proprietary formula that sets up binding conditions for the QIAprep Miniprep column's silica-gel-membrane. Buffer P3 sets up binding conditions for QIAGEN anion-exchange columns. Our website explains QIAGEN's nucleic acid purification technologies in more detail.
FAQ ID -310
Do you have a protocol for the isolation of plasmid DNA from Proteus spp.?

Yes, please follow the User-Developed Protocol 'Isolation of plasmid DNA from Proteus spp. using the QIAGEN Plasmid Midi Kit' (QP09).

FAQ ID -889
Do you have a protocol for the isolation of plasmid DNA from Corynebacterium glutamicum?
FAQ ID -886
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 recipe for SOC medium?

The components of the SOC medium are:

  • 0.5% Yeast Extract
  • 2% Tryptone
  • 10 mM NaCl
  • 2.5 mM KCl
  • 10 mM MgCl2
  • 10 mM MgSO4
  • 20 mM Glucose*

*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.

FAQ ID -798
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
Do you have a protocol for the isolation of plasmid DNA from Oligotropha carboxidovorans?

Yes, please follow the User-Developed Protocol 'Isolation of plasmid from Oligotropha carboxidovorans using the QIAGEN Plasmid Midi Kit' (QP08).

FAQ ID -888
How should QIAGEN Plasmid Purification Kits be stored and for how long?
QIAGEN-tips and QIAfilter Cartridges should be stored dry and at room temperature (15–25°). They can be stored for at least two years without showing any reduction in performance, capacity, or quality of separation.

QIAGEN, QIAfilter, EndoFree, HiSpeed and Large-Construct Plasmid Kits should be stored at room temperature (15–25°). After adding RNase A, Buffer P1 should be stored at 2–8°C and is stable for six months. All other buffers and RNase A stock solution can be stored for two years at room temperature (15–25°).
FAQ ID -192
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
Do you have a protocol for the isolation of bacteriophage P1 derived constructs with any of your plasmid kits?

Yes, please follow the User-Developed Protocol 'Isolation of bacteriophage P1 derived constructs using the QIAGEN Plasmid Midi Kit' (QP03).

FAQ ID -883
Do you have a protocol for the isolation of single-stranded DNA from M13 phage using QIAGEN Plasmid Kits?

Yes, please follow the Supplementary Protocol 'Isolation of single-stranded DNA from M13 phage using QIAGEN Plasmid Kits' (QP14).

FAQ ID -894
How can I prevent clogging of QIAfilter cartridges?
It is important to completely mix and lyse bacterial cells with plasmid Buffers P1 and P2. Incomplete mixing results in sticky or slimy areas of lysate, which will clog the filter matrix. It is recommended to completely resuspend cells in Buffer P1 and vigorously mix after addition of Buffer P2. Also mixing after Buffer P3 addition needs to be complete to allow fluffy precipitation of cell debris, which will float up. If the white debris does not float, dislodge it from the QIAfilter barrel wall (e.g. using a sterile pipette tip). Otherwise it will collect on the filter matrix and can lead to clogging. Use of LyseBlue reagent will help to achieve proper mixing results.
FAQ ID -1060
What are the maximum culture volumes to use with the QIAGEN Plasmid Midi or Maxi Kit?

Maximum recommended culture volumes for standard Luria Bertani (LB) medium*:

 

 

QIAGEN-tip 100

(QIAGEN Plasmid Midi Kit)

QIAGEN-tip 500

(QIAGEN Plasmid Maxi Kit)

High-copy plasmids

25 ml 100 ml

Low-copy plasmids

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.

FAQ ID -167
What is the composition of buffer QBT?

Buffer QBT is the equilibration buffer used in QIAGEN Plasmid Kits for plasmid purification and in QIAGEN Blood & Cell culture kits.

The composition of Buffer QBT is:

  • 750mM NaCl • 50 mM MOPS, pH 7.0
  • 15% isopropanol (v/v)
  • 0.15 % Triton® X-100 (v/v)

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

FAQ ID -411
When is chloramphenicol amplification of plasmids performed?

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.

FAQ ID -3
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