EndoFree Plasmid Kits

Für die Aufreinigung von bis zu 10 mg endotoxinfreier Plasmid- oder Cosmid-DNA in hochwertiger Transfektionsqualität

S_2544_ADNA_EndoFreePLS

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EndoFree Plasmid Maxi Kit (10)

Cat. No. / ID:   12362

10 QIAGEN-tip 500, Reagenzien, 10 QIAfilter Maxi Cartridges, endotoxinfreie Puffer
452,00 €
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KitPuffer
EndoFree Plasmid Kit
EndoFree Plasmid Buffer Set
Kartuschentyp
Maxi
Mega
Giga
EndoFree Plasmid Kits sind für molekularbiologische Anwendungen vorgesehen. Diese Produkte sind nicht zur Diagnose, Prävention oder Behandlung einer Erkrankung vorgesehen.

✓ Automatische Verarbeitung von Online-Bestellungen 24/7

✓ Sachkundiger und professioneller technischer und Produkt-Support

✓ Schnelle und zuverlässige (Nach-)Bestellung

Eigenschaften

  • Weniger als 0,1 EU/µg erhaltener DNA
  • Integrierte Endotoxinentfernung
  • Hohe Ausbeute an Plasmid-DNA mit hoher Kopienzahl
  • LyseBlue für optimale Lyse und maximale DNA-Ausbeute

Angaben zum Produkt

EndoFree Plasmid Kits ermöglichen die schnelle, auf Anionenaustausch basierende endotoxinfreie Aufreinigung von Plasmid-DNA. QIAfilter Cartridges ermöglichen eine schnelle Lysatbereinigung durch Filtration. Die aufgereinigte DNA übertrifft die durch zweimalige CsCl-Gradientenzentrifugation erzielte Reinheit und eignet sich für anspruchsvolle Transfektionsanwendungen. Das EndoFree Plasmid Buffer Set kann zum Ansetzen von Plasmid- oder Cosmid-DNA-Präparationen in Transfektionsqualität von 10 Mega oder 5 Giga genutzt werden.

Leistung

Die EndoFree Plasmid Kits enthalten im Plasmid-Aufreinigungsverfahren einen effizienten Schritt zur Endotoxinentfernung — zur Entfernung von Lipopolysacchariden werden keine zusätzlichen Extraktionen oder Affinitätssäulen benötigt. Bakterienlysate werden durch Filtration mit einer QIAfilter Mega-Giga oder Maxi Kartusche bereinigt, und Plasmid-DNA wird mittels QIAGEN-Tips mit Anionenaustauscherharz, die nach dem Schwerkraftprinzip arbeiten, aufgereinigt. Die Ausbeute aus der Kultur beträgt bis zu 10 mg (Giga), 2,5 mg (Mega) bzw. 500 μg (Maxi) aufgereinigter DNA (das Kulturvolumen hängt von der Plasmidkopienzahl, Insertgröße, dem Wirtsstamm und dem Kulturmedium ab). Die aufgereinigte DNA ist endotoxinfrei (<0,1 EU/µg DNA).

Für die Aufreinigung von Low-Copy-Plasmiden und -Cosmiden eignet sich das EndoFree Plasmid Mega Kit besser als das EndoFree Giga Plasmid Kit, da große Kulturvolumina benötigt werden und die Kapazität der QIAfilter Mega-Giga Cartridge begrenzt ist.

EndoFree Plasmid Kits entfernen die während der Lyse freigesetzten bakteriellen Endotoxine, welche die DNA-Transfektion in Primärzellen und sensitive Kulturzellen beeinflussen. Die aus den EndoFree Plasmid Kits gewonnene endotoxinfreie DNA eignet sich hervorragend für reproduzierbare und zuverlässige Transfektionsergebnisse (siehe Abbildungen „Zusammenhang zwischen Plasmidaufreinigungsverfahren und Transfektionseffizienz“ und „Zusammenhang zwischen Plasmidreinheit und Transfektionseffizienz“ sowie die Tabellen „Endotoxingehalt in Plasmidpräparationen“ und „EndoFree-DNA liefert hohe Transfektionseffizienz in Primärzellen“). Die hochreine endotoxinfreie DNA von QIAGEN ist auch für die Gentherapieforschung und andere anspruchsvolle Anwendungen geeignet.

Endotoxinspiegel in Plasmidpräparationen*
Verfahren der Plasmidpräparation Endotoxin
(EU/µg DNA)
Mittlere Transfektions
effizienz
EndoFree Plasmid Kit 0,1 154%
QIAGEN Plasmid Kit 9,3 100%
2x CsCl 2,6 99%
Silikagel-Aufschlämmung 1230,0 24%
EndoFree-DNA liefert hohe Transfektionseffizienz in Primärzellen*
DNA-Aufreinigungsverfahren Prozentsatz transfizierter Zellen
EndoFree Plasmid Kit 21,0% ± 0,93
QIAGEN Plasmid Kit 8,1% ± 0,57
Silikagel-Aufschlämmung 5,2% ± 0,74

Prinzip

Der Grad der Endotoxin-Kontamination in aufgereinigter Plasmid-DNA hängt vom verwendeten Aufreinigungsverfahren ab (siehe Tabelle „Endotoxingehalt in Plasmidpräparationen“). Mit Silika aufgeschlämmte DNA weist extrem hohe Endotoxinwerte auf. QIAGEN, QIAfilter und HiSpeed Plasmid Kits sowie zweimalige CsCl-Ultrazentrifugation ergeben sehr reine DNA mit relativ geringen Endotoxinwerten. Um Plasmid-DNA mit einem Reinheitsgrad von <0,1 EU/µg Plasmid-DNA zu erhalten, umfassen die EndoFree Plasmid Kits einen integrierten Schritt zur Endotoxinentfernung.

Die in den QIAfilter, HiSpeed und EndoFree Plasmid Kits mitgelieferten QIAfilter Cartridges sind spezielle Filtereinheiten und ersetzen die Zentrifugation nach der alkalischen Lyse von Bakterienzellen. QIAfilter Cartridges entfernen SDS-Präzipitate vollständig und bereinigen bakterielle Lysate in einem Bruchteil der für die Zentrifugation benötigten Zeit, wodurch sich die Dauer der Plasmidaufreinigung um bis zu 1 Stunde verkürzt. QIAfilter Mega-Giga Cartridges nutzen das hausinterne Vakuum, um auch große Mengen an Bakterienlysat mit minimalem Aufwand effizient zu bereinigen (bitte beachten, dass die Flasche nicht im Lieferumfang der Kits enthalten ist). QIAfilter Maxi Cartridges haben ein Spritzenformat und die Lysate werden in Sekundenschnelle bereinigt, indem die Flüssigkeit durch den Filter gedrückt wird.

Das innovative Anionenaustauscherharz in den QIAGEN-tips wurde ausschließlich für die Aufreinigung von Nukleinsäuren entwickelt. Seine hervorragenden Trenneigenschaften führen zu einer DNA-Reinheit, die der durch zwei aufeinanderfolgende Durchgänge der CsCl-Gradientenzentrifugation erzielten Reinheit gleichwertig oder überlegen ist. Vorgepackte QIAGEN-Spitzen arbeiten nach dem Schwerkraftprinzip und trocknen nie aus, so dass der Zeitaufwand für das manuelle Ansetzen von Plasmiden minimiert wird. Im gesamten QIAGEN Plasmid-Aufreinigungssystem kommen keine toxischen Substanzen wie Phenol, Chloroform, Ethidiumbromid und CsCl zum Einsatz, was die Gefahr für den Anwender und die Umwelt minimiert.

Endotoxine, auch bekannt als Lipopolysaccharide oder LPS, sind Bestandteile der Zellmembran gramnegativer Bakterien wie E. coli (siehe Abbildung „ Bakterielle Zellwand“). Während des Lyse-Schrittes der Plasmidaufreinigung werden Endotoxine freigesetzt, die die Transfektionseffizienz bei Endotoxin-empfindlichen Zelllinien erheblich verringern (siehe Abbildungen „ Zusammenhang zwischen Plasmidaufreinigungsverfahren und Transfektionseffizienz“ und „ Zusammenhang zwischen Plasmidreinheit und Transfektionseffizienz“ sowie die Tabellen „Endotoxingehalte in Plasmidpräparationen“ und „EndoFree DNA liefert hohe Transfektionseffizienz in Primärzellen“). Außerdem können Endotoxine die Aufnahme von Plasmid-DNA in Transfektionsexperimenten beeinflussen, indem sie mit der DNA um “freies” Transfektionsreagenz konkurrieren. Endotoxine lösen auch eine unspezifische Aktivierung von Immunreaktionen in Immunzellen wie Makrophagen und B-Zellen aus, was zu einer Fehldeutung der Transfektionsergebnisse führen kann. Zu diesen Folgen gehört die induzierte Synthese von Proteinen und Lipiden wie IL-1 und Prostaglandin. Insgesamt stellen Endotoxine beim Aufbau von Transfektionsexperimenten eine nicht beeinflussbare Variable dar, die das Resultat und die Reproduzierbarkeit der Ergebnisse beeinflusst und deren Vergleich und Interpretation erschwert. In der Gentherapieforschung können Endotoxine durch Auslösung des endotoxischen Schocksyndroms und Aktivierung der Komplementkaskade Störeinflüsse verursachen.

Spezifikationen

Merkmale
EndoFree Plasmid
Maxi Kit
EndoFree Plasmid
Mega Kit
EndoFree Plasmid
Giga Kit
Anwendungen Forschung zur Gentherapie, Transfektion sensitiver Zellen Forschung zur Gentherapie, Transfektion sensitiver Zellen Forschung zur Gentherapie, Transfektion sensitiver Zellen
Kulturvolumen/Ausgangsmaterial 100–250 ml Kulturvolumen 500 ml – 2,5 Liter Kulturvolumen 2,5 Liter Kulturvolumen
Plasmidtyp High-Copy, Low-Copy, Cosmid-DNA High-Copy, Low-Copy, Cosmid-DNA High-Copy, Low-Copy, Cosmid-DNA
Verfahren Manuell (Schwerkraftprinzip) Manuell (Schwerkraftprinzip) Manuell (Schwerkraftprinzip)
Probe pro Lauf 1 Probe pro Lauf 1 Probe pro Lauf 1 Probe pro Lauf
Dauer pro Lauf 150 min 220 min 310 min
Ausbeute <500 µg <2,5 mg <10 mg
Abbildungen ansehen

Verfahren

Die Bakterienzellen werden unter alkalischen Bedingungen lysiert und die Rohlysate mit der QIAfilter Cartridge bereinigt. Anschließend wird dem gefilterten Lysat der Puffer zur Endotoxinentfernung zugesetzt und das Lysat auf Eis inkubiert. Das bereinigte Lysat wird dann auf die Anionenaustauscher-Spitze aufgebracht, wo die Plasmid-DNA unter geeigneten salzarmen und pH-Bedingungen selektiv bindet. RNA, Proteine, Metaboliten und andere niedermolekulare Verunreinigungen werden durch einen Waschvorgang mit mittlerem Salzgehalt entfernt, und hochreine Plasmid-DNA wird in einem Puffer mit hohem Salzgehalt eluiert (siehe Flussdiagramm „ QIAGEN Plasmid Kit Verfahren“). Die DNA wird konzentriert, durch Isopropanol-Fällung entsalzt und durch Zentrifugation gesammelt.

Abbildungen ansehen

Anwendungen

Die mit den EndoFree Plasmid Kits aufgereinigte DNA eignet sich für alle anspruchsvollen Anwendungen einschließlich:

  • Transfektion, einschließlich primärer, sensitiver und Suspensionszellen
  • Gentherapie-Forschung
  • Gen-Silencing
  • Mikroinjektion

Ergänzende Daten und Abbildungen

Ressourcen

Brochures & Guides (1)
EndoFree® Kits
PDF (103KB)
Eco-friendlier* and quicker plasmid DNA preparation for transfection
Quick-Start Protocols (2)
Safety Data Sheets (1)
Supplementary Protocols (2)
This protocol is for purification of up to 100 µg endotoxin-free plasmid DNA using QIAGEN-tip 100.
Endotoxin-free DNA is essential for gene therapy research and will improve transfection into sensitive eukaryotic cells.
Technical Information and Important Notes (1)
Certificates of Analysis (1)

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
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
Which QIAGEN plasmid preparation kits will contain LyseBlue Reagent?

LyseBlue reagent is provided in the following QIAGEN plasmid kits:

FAQ ID -865
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
What is the composition of buffer QN?

The composition of Buffer QN is:

  • 1.6 M NaCl
  • 50 mM MOPS, pH 7.0
  • 15 % isopropanol (v/v)

Buffer QN is the elution buffer used in EndoFree Plasmid Kits for preparation of endotoxin-free plasmid DNA. Details on buffer preparation and storage are presented in Appendix C of the EndoFree Plasmid Purification Handbook.

FAQ ID -414
3299-Can Buffer ER from the Endofree Plasmid kits be used instead of the Buffer ETR in QIAGEN Plasmid Plus kits?

No, the two buffers cannot be interchanged.  The QIAGEN Plasmid Plus chemistry is not compatible with Buffer ER from the Endofree Plasmid kits.

Which QIAGEN kit do you recommend for purifying plasmid DNA suitable for transfection of sensitive cells?
We recommend our EndoFree Plasmid Kits to isolate plasmid DNA suitable for transfecting sensitive cells and primary cells. The Endofree Plasmid kits are designed to remove endotoxins (i.e. lipopolysaccharides that are part of the bacterial cell wall) and are generally advantageous in providing higher transfection efficiencies.
FAQ ID -1092
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
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
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
What is the composition of Buffer ER? Is it available separately?
The composition of buffer ER is confidential. It is included in the EndoFree Plasmid Kits, and it is not sold separately.
FAQ ID -571
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
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 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
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
How can I keep my centrifuge tubes endotoxin-free?

In order to avoid recontamination of plasmid DNA after initial endotoxin removal, we recommend using only new plasticware which is certified to be pyrogen- or endotoxin-free. Endotoxin-free or pyrogen-free plasticware can be obtained from many different suppliers. Endotoxins adhere strongly to glassware and are difficult to remove completely during washing. Standard laboratory autoclaving procedures have little or no effect on endotoxin levels. Moreover, if the autoclave has previously been used for bacteria, the glassware will become extensively contaminated with endotoxin molecules. Heating glassware at 180°C overnight is recommended to destroy any attached endotoxin molecules. For further reading on endotoxin removal, please refer to the scientific literature (e.g. Weary M. and Pearson F., 1988. A manufacturer's guide to depyrogenation. Biopharm 1, 22-29).

General information on endotoxins, which can be efficiently eliminated with QIAGEN's EndoFree Plasmid Kits, is available online at our Plasmid Resource Center.

FAQ ID -301
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
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 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