EndoFree Plasmid Kits

10 mgまでのエンドトキシンフリーなトランスフェクショングレードのプラスミドおよびコスミドDNA精製用

S_2544_ADNA_EndoFreePLS

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EndoFree Plasmid Maxi Kit (10)

Cat. No. / ID:  12362

10 QIAGEN-tip 500, Reagents, 10 QIAfilter Maxi Cartridges, Endotoxin-free Buffers.
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KitBuffer
EndoFree Plasmid Kit
EndoFree Plasmid Buffer Set
Cartridge type
Maxi
Mega
Giga
EndoFree Plasmid Kitsは分子生物学的アプリケーション用であり、疾病の診断、予防、あるいは治療に使用することはできません。

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Features

  • プラスミドDNA 1 µg あたり0.1 EU 以下
  • エンドトキシン除去ステップを組み込んだ迅速な操作手順
  • 高い収量の高コピープラスミドDNA
  • LyseBlue試薬により最適な溶解操作と最大のDNA収量

Product Details

EndoFree Plasmid Kitsは陰イオン交換をベースにしたエンドトキシンフリーのプラスミドDNA精製を迅速に行ないます。QIAfilter Cartridgesはろ過による迅速なライセート清澄化を実現します。精製したDNA は、CsCl 密度勾配遠心法を2 回行なって得られる精製グレードに匹敵し、高感度トランスフェクション等のアプリケーションに最適です。EndoFree Plasmid Buffer Setは、10 megaまたは5 gigaのトランスフェクショングレードのプラスミドまたはコスミドDNA調製に使用できます。

Performance

EndoFree Plasmid Kitsには、リポ多糖類を除去するために追加の抽出ステップやアフィニティーカラムは不要で、プラスミド精製操作工程に効率的なエンドトキシン除去ステップが組み込まれています。QIAfilter Mega-GigaまたはMaxi Cartridgeによるろ過操作でバクテリアライセートを清澄化し、陰イオン交換樹脂の含有されたオープンカラム方式のQIAGEN-tips でプラスミドDNA を精製します。最大10 mg(Giga)、2.5 mg(Mega)、500 µg(Maxi)のDNAが培養液から精製されます(培養液量はプラスミドのコピー数、添加サイズ、宿主株、培養媒体に依存します)。精製したDNAは、エンドトキシンフリーです(0.1 EU/µg 以下のDNA)。

低コピー数のプラスミド、コスミドの精製では、必要な培養液量が多く、QIAfilter Mega-Giga Cartridgeの容量は制限があるため、EndoFree Plasmid Giga KitよりEndoFree Plasmid Mega Kitの使用をお薦めします。

EndoFree Plasmid Kitsは、溶解ステップ中に放出され、初代細胞や感受性の高い細胞株へのトランスフェクションに影響を与えるバクテリアのエンドトキシンを除去します。EndoFree Plasmid Buffer Setを用いて得られたエンドトキシンを含まないDNAは、トランスクフェション実験に最適で、再現性が高く信頼できる結果が得られます(図“ プラスミド精製法とトランスフェクション効率の比較”および“ プラスミド純度とトランスフェクション効率の比較”、表 "異なるプラスミド調製法によるエンドトキシンレべルの比較”および“EndoFree DNAで初代培養細胞へ高いトランスフェクション効率)。QIAGENの超高純度なエンドトキシン・フリーなDNAは遺伝子治療研究やその他の高感度なアプリケーションに最適です。

異なるプラスミド調製法によるエンドトキシンレべルの比較*
プラスミド調製法 エンドトキシン
(EU/µg DNA)
トランスフェクション
効率の平均
EndoFree Plasmid Kit 0.1 154%
QIAGEN プラスミド調製用キット 9.3 100%
2x CsCl 2.6 99%
シリカゲル懸濁液 1230.0 24%
* 宿主菌株:DH5αプラスミド:pRSVcat.
1 ng LPS = 1.8 EU.
図 " プラスミド精製法とトランスフェクション効率の比較"のデータから算出。
EndoFree DNAで初代培養細胞へ高いトランスフェクション効率
DNA 精製法 トランスフェクトされた細胞の割合
EndoFree Plasmid Kit 21.0% ± 0.93
QIAGEN プラスミド調製用キット 8.1% ± 0.57
シリカゲル懸濁液 5.2% ± 0.74
* 表記の方法でpEGFP-N2 (CLONTECH) を調製し、初代ウサギ胃壁培養細胞にトランスフェクトした。トランスフェクションには、Effectene Transfection Reagentを使用した。48時間後にGFPの発現細胞数からトランスフェクトされた細胞の割合を決定した。トランスフェクション効率は、各精製法で少なくとも2回のDNA精製を行ない、6回から9回のトランスフェクションから得た結果の平均値を表示した。(データ提供:C. Chew and J. Parente, Department of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia, USA.)
See figures

Principle

精製したプラスミドDNA中のエンドトキシンの混入レベルは用いた精製法に依存します(表 “異なるプラスミド調製法によるエンドトキシンレべルの比較”)。シリカ懸濁液で精製したDNAは、非常に高いエンドトキシンレベルを示します。QIAGEN Plasmid Kits、QIAfilter およびHiSpeed Plasmid Kits そして2回のCsCl 密度勾配遠心分離法で調製した高純度のDNAは、比較的低いレベルのエンドトキシンしか含んでいません。エンドトキシン除去ステップが組み込まれたEndoFree Plasmid Buffer Setでは、プラスミドDNA 1 μg当たり0.1 EU以下のエンドトキシン含有量のプラスミドDNAが得られます。

QIAfilter Cartridges(EndoFree Plasmid Buffer Setには付属していない)は、バクテリア細胞のアルカリ溶解後に行なう遠心操作に代わる方法として開発された特別なフィルターです。QIAfilter CartridgesはSDS沈殿物を完全に取り除くばかりではなく、遠心操作に必要な時間よりも短時間でバクテリアライセートの清澄化を可能にし、プラスミド精製時間を最大1時間短縮できます。QIAfilter Cartridges(EndoFree Plasmid Buffer Setには付属していない)はバクテリアライセートを簡単かつ効率的に清澄化します。QIAfilter Maxi Cartridgesは、シリンジフォーマットで、液体を注入することにより溶解物がフィルターを通過し、すばやく清澄化されます。

QIAGEN-tip(EndoFree Plasmid Buffer Setには付属していない )中の非常にユニークな陰イオン交換樹脂は核酸精製を目的として開発されました。本製品の優れた核酸分離特性により、CsCl 密度勾配遠心分離法を2回行なって得たDNAの純度に匹敵、あるいはそれ以上の純度のDNAが調製されます。充填済みQIAGEN-tipsはオープンカラムで操作し、乾燥することはなく、プラスミド調製に必要なマニュアルでの作業時間を短縮できます。全てのQIAGENプラスミド精製システムでは、ユーザーおよび環境への危害が最小限となるように、フェノール、クロロホルム、臭化エチジウム、CsCl等の有害な試薬を一切使用していません。

リポ多糖類あるいはLPSとして知られているエンドトキシンはE. coli のようなグラム陰性菌の細胞膜構成成分です(図 “ バクテリア細胞壁”)。エンドトキシンはプラスミド精製の溶解ステップ中に放出され、エンドトキシン感受性細胞株へのトランスフェクション効率を顕著に低下させます(図“ プラスミド精製法とトランスフェクション効率の比較”および“ プラスミド純度とトランスフェクション効率の比較”)。さらにエンドトキシンはDNAと結合していない“フリー”のトランスフェクション試薬と競合することにより、トランスフェクション実験におけるプラスミドDNAの取り込みに影響を与えます。エンドトキシンの混在はまた、マクロファージやB 細胞のような免疫系細胞における免疫応答を非特異的に活性化するため、間違ったトランスフェクション解釈につながることもあります。これらの応答には、IL-1 やプロスタグランジン等のタンパク質や脂質の生産誘導も含まれています。全エンドトキシンの混入はトランスフェクション実験のセットアップにおいてコントロールできない変動要素となるため、実験結果およびその再現性に影響し、結果の比較および解釈が困難になります。さらに、エンドトキシンショック症候群や、補体系カスケードの活性化を導くエンドトキシンは、遺伝子治療に大きな影響を与えます。

製品仕様

特徴
EndoFree Plasmid
Giga Kit 
EndoFree Plasmid
Mega Kit
EndoFree Plasmid
Maxi Kit
Applications Research on gene therapy, transfection of sensitive cells Research on gene therapy, transfection of sensitive cells Research on gene therapy, transfection of sensitive cells
Culture volume/starting material 2.5 liters culture volume 500 ml – 2.5 liters culture volume 100–250 ml culture volume
Plasmid type High-copy, low-copy, cosmid DNA High-copy, low-copy, cosmid DNA High-copy, low-copy, cosmid DNA
Processing Manual (vacuum) Manual (vacuum and centrifugation) Manual (vacuum)
Sample per run 1 sample per run 1 sample per run  1 sample per run 
Time per run 310 min 220 min  150 min
Yield <10 mg <2.5 mg <500 µg

See figures

Procedure

バクテリア細胞はアルカリ性条件下で溶解され、粗ライセートはQIAfilter Cartridgeを用いて清澄化されます。 この段階でEndotoxin Removal Bufferをろ過したライセートに添加し、氷上でインキュベートします。清澄化したライセートを陰イオン交換チップ上にアプライすると、適切な低塩濃度あるいはpH条件でプラスミドDNAが選択的に結合します。RNA、タンパク質、二次代謝物、その他の低分子量不純物が、中濃度の塩による洗浄で取り除かれ、超高純度プラスミドDNAが高塩濃度のバッファーで溶出されます(フローチャート“ QIAGEN Plasmid Kit 操作手順の比較”)。イソプロパノール沈殿によりDNAが濃縮、脱塩され、遠心操作により回収されます。

See figures

Applications

EndoFree Plasmid Kitsで精製したDNAは以下のような高感度なアプリケーションに最適です。

  • 初代細胞、感受性の高い細胞や懸濁細胞へのトランスフェクション
  • 遺伝子治療研究
  • 遺伝子サイレンシング
  • マイクロインジェクション

Supporting data and figures

Resources

テクニカルインフォメーション (1)
クイックスタートプロトコール (2)
キットハンドブック (1)
MSDS (1)
Download Safety Data Sheets for QIAGEN product components.
Supplementary Protocols (2)
Endotoxin-free DNA is essential for gene therapy research and will improve transfection into sensitive eukaryotic cells.
This protocol is for purification of up to 100 µg endotoxin-free plasmid DNA using QIAGEN-tip 100.
Certificates of Analysis (1)
Quick-Start Protocols (2)

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