QIAGEN Plasmid Kits pour l’extraction de l’ADN plasmidique

Pour la purification de 10 mg maximum d’ADN plasmidique ou de cosmide de qualité de transfection

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QIAGEN Plasmid Mini Kit (25)

N° de réf. / ID. 12123

25 QIAGEN-tip 20, réactifs, tampons

KitTamponColonneCartouche

Plasmid Kit

Plasmid Buffer Set

QIAGEN-tip

QIAfilter Cartridge

Type de colonne

Mini

Midi

Maxi

Mega

Giga

Préparations

100

Sur demande

Les QIAGEN Plasmid Kits sont destinés aux applications de biologie moléculaire. Ces produits ne sont pas conçus pour le diagnostic, la prévention ou le traitement des maladies.

✓ Traitement automatique des commandes en ligne 24 h/24 7 j/7

✓ Assistance technique et produits pertinente et professionnelle

✓ Commande (ou réapprovisionnement) rapide et fiable

Caractéristiques

Pureté égale à celle de la double centrifugation en gradient de CsCl
Hauts rendements d’ADN plasmidique
Préparations économiques
LyseBlue pour lyse optimale et rendement d’ADN maximal

Détails produit

Les QIAGEN Plasmid Kits fournissent des embouts d’écoulement par gravité et échangeurs d’anions pour la purification de l’ADN plasmidique de qualité de transfection. Le nettoyage des lysats et la précipitation à l’isopropanol sont réalisés par centrifugation.

Le QIAGEN Plasmid Mega Kit (n° de réf. 12181) et le QIAGEN Plasmid Giga Kit (n° de réf. 12191) peuvent être utilisés avec les QIAfilter Mega-Giga Cartridges (n° de réf. 19781) dans le cadre d’une étape facultative du protocole pour un nettoyage rapide des lysats bactériens par filtration plutôt que par centrifugation. Plus d’informations concernant le principe sont disponibles ci-dessous.

Performances

Les QIAGEN Plasmid Kits utilisent des embouts QIAGEN d’écoulement par gravité et échangeurs d’anions pour la purification efficace de l’ADN plasmidique. Une quantité maximale de 10 mg (giga), 2,5 mg (méga), 500 µg (maxi), 100 µg (midi) et 20 µg (mini) d’ADN plasmidique à nombre de copies élevé est purifiée à partir de la culture (les volumes de culture dépendent du nombre de copies plasmidiques, de la taille de l’insert, de la souche hôte et du milieu de culture). L’ADN plasmidique purifié grâce aux QIAGEN Plasmid Kits convient parfaitement pour une utilisation dans des applications telles que la transfection (voir la figure « Efficacité de la transfection par rapport à la méthode de purification des plasmides »), le clonage et la transcription in vitro.

Principe

La résine échangeuse d’anions unique des QIAGEN-tips est élaborée exclusivement pour la purification des acides nucléiques. Ses propriétés de séparation exceptionnelles produisent un ADN dont la pureté est égale ou supérieure à celui obtenu par deux cycles successifs de centrifugation en gradient de CsCl. Les QIAGEN-tips préemballés fonctionnent par écoulement par gravité et ne s’assèchent jamais, ce qui réduit le temps de manipulation nécessaire à la préparation des plasmides. L’intégralité du système de purification de plasmides QIAGEN évite l’utilisation de substances toxiques telles que le phénol, le chloroforme, le bromure d’éthidium et le CsCl, en réduisant au minimum les dangers pour l’utilisateur et l’environnement.

Dans le cadre d’une étape facultative du protocole pour le nettoyage rapide des lysats bactériens par filtration plutôt que par centrifugation, vous pouvez utiliser les QIAfilter Mega-Giga Cartridges (n° de réf. 19781) qui fonctionnent avec du vide interne pour nettoyer efficacement même les grands volumes de lysats bactériens avec un minimum d’effort. Le protocole est disponible dans le manuel.

Caractéristiques
Fonctionnalités	Plasmid Mini Kit	Plasmid Midi Kit	Plasmid Maxi Kit	Plasmid Mega Kit	Plasmid Giga Kit
Applications	Transfection, clonage, séquençage, séquençage capillaire, etc.	Transfection, clonage, séquençage, séquençage capillaire, etc.	Transfection, clonage, séquençage, séquençage capillaire, etc.	Transfection, clonage, séquençage, séquençage capillaire, etc.	Transfection, clonage, séquençage, séquençage capillaire, etc.
Volume de culture/matériel de départ	Volume de culture de 3 à 10 ml	Volume de culture de 25 à 100 ml	Volume de culture de 100 à 500 ml	Volume de culture de 500 ml à 2,5 l	Volume de culture de 2,5 à 5 l
Volume d’élution	Variable	Variable	Variable	Variable	Variable
Type de plasmide	ADN de cosmide à nombre de copies élevé, à nombre de copies faible	ADN de cosmide à nombre de copies élevé, à nombre de copies faible	ADN de cosmide à nombre de copies élevé, à nombre de copies faible	ADN de cosmide à nombre de copies élevé, à nombre de copies faible	ADN de cosmide à nombre de copies élevé, à nombre de copies faible
Traitement	Manuel (écoulement par gravité)	Manuel (écoulement par gravité)	Manuel (écoulement par gravité)	Manuel (écoulement par gravité)	Manuel (écoulement par gravité)
Échantillon par cycle d’exécution	1 échantillon par cycle d’exécution	1 échantillon par cycle d’exécution	1 échantillon par cycle d’exécution	1 échantillon par cycle d’exécution	1 échantillon par cycle d’exécution
Technologie	Technologie d’échange d’anions	Technologie d’échange d’anions	Technologie d’échange d’anions	Technologie d’échange d’anions	Technologie d’échange d’anions
Durée par cycle d’exécution	80 min	150 min	160 min	220 min	320 min
Rendement	< 20 µg	jusqu’à 100 µg	< 500 µg	< 2,5 mg	< 10 mg

Procédure

Avec les QIAGEN Plasmid Kits, les lysats bactériens sont nettoyés par centrifugation. Le lysat nettoyé est ensuite chargé sur l’embout échangeur d’anions, là où l’ADN plasmidique se lie de façon sélective dans des conditions appropriées de faible salinité et de pH. L’ARN, les protéines, les métabolites et d’autres impuretés à faible masse moléculaire sont éliminés par un lavage moyennement salé et l’ADN plasmidique pur est élué dans un tampon à salinité élevée (voir le schéma « Procédures QIAGEN Plasmid Kit »). L’ADN est concentré et dessalé par une précipitation à l’isopropanol et collecté par centrifugation.

Applications

L’ADN plasmidique purifié grâce aux QIAGEN Plasmid Kits convient parfaitement pour une utilisation dans des applications telles que :

Transfection
Clonage
PCR
Transcription in vitro

Données et illustrations utiles

Efficacité de la transfection par rapport à la méthode de purification de plasmides.

Différentes préparations d’ADN contenant le pRSVcat et utilisant les méthodes énoncées ont été introduites dans les lignées cellulaires indiquées par transfection par liposomes, et les efficacités ont été déterminées par la mesure des niveaux d’expression du gène CAT après 40 h. Chaque barre représente la moyenne de quatre transfections indépendantes (deux transfections avec chacune deux préparations de plasmides indépendantes). L’efficacité de transfection la plus élevée a été obtenue avec les QIAGEN Plasmid Kits.

Spécifications

Caractéristiques	Spécifications
Technology	Technologie d’échange d’anions
Culture volume/starting material	Volume de culture de 3 ml à 5 l
Yield	< 20 µg à < 10 mg
Processing	Manuel (écoulement par gravité)
Samples per run (throughput)	1 échantillon par cycle d’exécution
Time per run or prep per run	80 à 320 min
Applications	Transfection, clonage, séquençage, séquençage capillaire, etc.
Plasmid type	ADN de cosmide à nombre de copies élevé, à nombre de copies faible

Ressources

April 2012

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.

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.

Download Safety Data Sheets for QIAGEN product components.

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

High-level carbapenem resistance in a Klebsiella pneumoniae clinical isolate is due to the combination of bla(ACT-1) beta-lactamase production, porin OmpK35/36 insertional inactivation, and down-regulation of the phosphate transport porin phoe.

Kaczmarek FM; Dib-Hajj F; Shang W; Gootz TD;

Antimicrob Agents Chemother; 2006; 50 (10):3396-406 2006 Oct PMID:17005822

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

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-1

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-1031

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-1045

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-1059

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-167

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-168

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-192

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-198

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-203

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-212

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-213

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-3

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 h to precipitate the DNA
Recover the precipitated DNA by centrifugation at full speed in a microcentrifuge for 15–20 min
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-305

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-3082

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-310

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-350

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-352

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-353

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-366

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-411

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-412

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-413

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-415

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-416

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-418

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-500

Redissolve the DNA by warming the solution slightly, e.g. incubate it at 37°C, and allow more time for redissolving.

FAQ-572

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-768

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-769

Yes, please follow the User-Developed Protocol:

'Isolation of plasmid DNA from yeast using the QIAGEN Plasmid Midi Kit' (QP11)

FAQ-853

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-859

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-861

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-862

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-864

LyseBlue reagent is provided in the following QIAGEN plasmid kits:

QIAGEN Plasmid Kits (Mini-, Midi-, Maxi-, Mega and Giga)
QIAfilter Plasmid Kits (Midi-, Maxi-, Mega and Giga)
HiSpeed Plasmid Kits (Midi- and Maxi)
EndoFree Plasmid Kit (Maxi-, Mega and Giga)
QIAprep Spin Miniprep Kit
QIAGEN Plasmid Plus Kits (Midi, Maxi, Mega and Giga)

FAQ-865

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-881

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

FAQ-883

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

FAQ-884

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

FAQ-885

Yes, please follow the User-Developed Protocol 'Isolation of Plasmid DNA from Corynebacterium glutamicum using the QIAGEN Plasmid Mini Kit' (QP06).

FAQ-886

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

FAQ-887

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

FAQ-888

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

FAQ-889

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

FAQ-890

Yes, please follow the User-Developed Protocol 'Isolation of very low-copy plasmids from Streptomyces spp. using the QIAGEN Plasmid Maxi Kit' (QP13).

FAQ-893

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

FAQ-894

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

FAQ-895

Yes, please follow either of the User-Developed Protocols:

FAQ-896

Yes, please follow the Supplementary Protocols:

FAQ-907

Yes, please follow the Supplementary Protocol 'Isolation of total RNA from plant tissue using the QIAGEN-tip' (QR01).

FAQ-949

TB Management

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Sexual & Reproductive Health

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Solutions for Laboratory-Developed Tests