QIAprep Spin Miniprep Kit

20 µgまでの分子生物学実験グレードのプラスミドDNA精製用

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QIAprep Spin Miniprep Kit (50)

Cat. No. / ID:  27104

For 50 high-purity plasmid minipreps: 50 QIAprep 2.0 Spin Columns, Reagents, Buffers, Collection Tubes (2 ml)
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KitColumn
QIAprep Spin Miniprep Kit
QIAprep 2.0 Spin Miniprep Columns
Eco-friendlier kit
Preparations
50
250
1000
QIAprep Spin Miniprep Kit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。
Is sustainability important to you?
For an eco-friendlier alternative to the selected product, look no further.

Features

  • 数分で即使用可能なプラスミドDNA調製
  • 再現性のある分子生物学実験グレードのプラスミドDNA収量
  • 高コピーと低コピーベクターの両方を調製できるプロトコール
  • High-Yield Supplementary Protocol によりさらに高い収量
  • 改善されたQIAprep 2.0 Spin Column
  • 便利なサンプル解析用GelPilotローディング色素

Product Details

QIAprep Spin Miniprep Kitは高純度なプラスミドDNAあるいはコスミドDNA(最高20 µg)を精製できます。精製DNAはシークエンシングおよびクローニングのようなルーチンの分子生物学アプリケーションに使用できます。High-Yield Supplementary Protocol により、さらに高い収量(最高30 µgまで)を得ることができます。


Performance

QIAprep Spin Miniprep Kitでは、分子生物学実験グレードのプラスミドDNAまたはコスミドDNA を最大20 µg 精製することができ、PCR、シークエンシング、クローニングのようなルーチンの分子生物学アプリケーションでの使用に適しています。汎用性のあるQIAprep 2.0 Spin Columns は、マイクロ遠心機または吸引マニホールドでき、QIAcube上で自動化も可能です(図 "QIAprep 2.0 Spin Columns操作オプション A B、および  C")。吸引法を使用することで、取り扱いが簡便になり、サンプル処理時間が短くなります。QIAprep 2.0 Spin Columns は、QIAvac 24 Plusをはじめとする市販のルアーコネクター付きマニホールドで吸引処理することができます。QIAprep Spin Miniprep Kit はQIAcube 上で自動化も可能です。

QIAprep Spin Miniprep Kitの詳細データ
フォーマット: スピンカラム
精製モジュール: QIAprep 2.0 Spin Columns
処理数: 1~24サンプル
操作時間: 30分で24ミニプレップ
必要な装置: マイクロ遠心分離機あるいは吸引装置;QIAcubeを用いて完全自動化
ライセート清澄化: 遠心操作
カラム容量 800 µl
最小溶出バッファー量 50 µl
高コピープラスミド用培養液量 1~500 ml
低コピープラスミド/コスミド用培養液量 1~10 ml

精製したDNAは、制限酵素分解で使用することができます(図 " 様々な制限酵素を用いた解析")。

See figures

Principle

QIAprep 2.0 Spin Columns は特殊なシリカメンブレンを有しています。 高濃度のカオトロピック塩の存在下で20 µg までのDNA がこのメンブレンに結合し、少量の低塩濃度のバッファーで溶出されます。QIAprepテクノロジーでは時間のかかるフェノール/クロロホルム抽出およびアルコール沈殿の必要がないだけではなく、樹脂漏れのリスクや、懸濁液取り扱いの難しさのような問題もありません。QIAprep 2.0 Spin Columns から溶出された高純度プラスミドDNA は、沈殿、濃縮、脱塩の必要がなく、即使用可能です。より速く、より便利にサンプル処理とその解析を可能にするゲルローディング色素がキットの中に含まれています。 GelPilot Loading Dyeは、アガロースゲル電気泳動操作の最適化を容易にし、より小さなDNAフラグメントをも監視するために、3種類ののトラッキング色素(キシレンシアノール、ブロモフェノールブルー、およびオレンジG)が含まれています(図 " GelPilot Loading Dye" を参照)。
See figures

Procedure

QIAprep Kit を用いたプラスミド精製法は、結合、洗浄そして溶出という非常にシンプルなものです(フローチャート" QIAprep操作手順" 参照)。まずバクテリアを溶解後、そのライセートを遠心操作により清澄化します。清澄化されたライセートをQIAprep 2.0モジュールにアプライし、プラスミドDNA をシリカメンブレンに結合します。夾雑物を洗い流し、高純度DNAだけを少量の溶出バッファーまたは水で溶出します。

QIAprep Kit は大腸菌からだけではなくビール酵母菌、枯草菌、Agrobacterium tumefaciensからのプラスミドDNA 精製にも利用できます。これらのプロトコールに関しては弊社テクニカルサポートにお問い合わせください。

See figures

Applications

QIAprep Miniprep Kit は、下記の用途を含むほとんどのアプリケーションに最適な高純度DNAを再現性良く調製します。

  • PCR
  • 制限酵素分解
  • ライゲーションやトランスフォーメーション
  • シークエンシング
  • スクリーニング

Supporting data and figures

Specifications

FeaturesSpecifications
ApplicationsFluorescent and radioactive sequencing (including capillary sequencing), ligation, cloning, transformation, etc.
ProcessingManual (vacuum or centrifugation) or automated (QIAcube Connect)
Plasmid typeHigh-copy, low-copy, cosmid DNA
Culture volume/starting material1–10 mL culture volume
Elution volume50 µl (minimal)
TechnologySilica technology
Time per run or prep per run<30 minutes
Yield<20 µg
Samples per run (throughput)1–24 samples per run
Number of preps per run1–24 samples per run

Resources

キットハンドブック (3)
アプリケーション/プロトコール (1)
For purification of up to 30 μg plasmid DNA
MSDS (1)
Download Safety Data Sheets for QIAGEN product components.
Supplementary Protocols (1)
Certificates of Analysis (1)
Application/Protocol Documents (1)
For purification of up to 30 μg plasmid DNA

FAQ

What is the composition of Buffer N3?
The composition of buffer N3 is confidential. It is a proprietary component of the QIAprep Spin Miniprep Kit. However, buffer N3 can be purchased separately. The catalogue number is 19064. The item is listed in the QIAGEN Product List online.
FAQ ID -767
How do I prepare Buffer MP?

Here is the protocol for preparing buffer MP:

 

  1. Dissolve 3.3 g citric acid monohydrate in 3 ml high-purity water at room temperature (21°C) in a 10 ml tube.
  2. Stir the solution at 200 rpm for 5 min.
  3. Filter the solution through a 0.2 μm sterile filter using a syringe to give a final volume of 6 ml Buffer MP.
FAQ ID - 3620
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
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
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
Are QIAprep and QIAquick Spin columns interchangeable?
No. While columns from the QIAprep Spin Miniprep Kit and the QIAquick PCR Purification- and Gel Extraction Kits are based on silica-gel-membrane technology, each is designed to work optimally within its own kit format. In addition, the binding capacity and DNA recovery size cut-offs of the QIAprep and QIAquick Spin columns are different. QIAprep Spin columns bind up to 20 ug of plasmid DNA up to 50 kb in length, while the QIAquick Spin columns bind up to 10 ug of DNA with a maximum fragment size of 10 kb.
FAQ ID -311
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 recommended culture medium for the QIAprep System?
Luria-Bertani (LB) broth is the recommended culture medium for use with QIAprep Kits, since richer broths such as TB (Terrific Broth) or 2x YT lead to extremely high cell densities, which can overload the purification system. Please review the section 'Culture Media' of Appendix A in the QIAprep Miniprep Handbook or visit our Plasmid Resource Center for additional information on optimal plasmid culturing and extraction conditions for all QIAGEN Plasmid Purification Kits.
FAQ ID -154
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
Can I use QIAprep Miniprep kits for low-copy plasmids and cosmids?

All QIAprep Miniprep Kits can be used for preparation of low-copy number plasmids and cosmids up to 50 kb. Below are recommendations for processing low-copy constructs using QIAprep technology:

  • Use up to 10 ml overnight E. coli cultures grown in LB medium
  • Be sure to include the optional Buffer PB wash step for all bacterial strains
  • When plasmids or cosmids are larger than 10 kb, pre-heat Buffer EB (or water) to 70°C prior to eluting DNA from the QIAprep membrane
  • When using 10 ml culture volume, it is recommended to double the volumes of Buffers P1, P2, and N3

See also QIAGEN News 1998, Issue 5 for an article entitled 'Isolation of a low-copy plasmid from agrobacterium using QIAprep technology'. Alternatively, the R.E.A.L. Prep 96 Plasmid Kit can be used for high-throughput purification of larger plasmids (e.g., BACs, PACs, and P1s). See QIAGEN News 1999, Issue 2 for an article entitled 'High-throughput purification of BACs with the new R.E.A.L. Prep 96 protocol'.

FAQ ID -127
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 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 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
What is the expected level of endotoxins in plasmid DNA purified with QIAprep spin miniprep kit?

QIAprep spin miniprep kit is based on silica extraction chemistry. Average endotoxin levels that we have observed for Silica gel slurry is around 1200 EU/µg DNA.

FAQ ID -3081
Do you have a protocol for the isolation of plasmid DNA from Bacillus subtilis?
Is it possible to elute plasmid DNA from the QIAprep Spin Miniprep columns with buffer containing Potassium Phosphate?

Unfortunately, we do not have any compatibility data for using potassium phosphate-based buffers instead of TE or water for the elution of plasmid DNA from the spin columns of the QIAprep Spin Miniprep Kit.

However, below is a reference where cDNA was eluted from QIAquick PCR Purification Kit columns with potassium phosphate buffer (4 mM, pH 8.5), after replacing the wash buffer (PE) with 5 mM potassium phosphate (pH 8.5) containing 80% ethanol:

Wang HY, Malek RL, Kwitek AE, Greene AS, Luu TV, Behbahani B, Frank B, Quackenbush J, Lee NH. "Assessing unmodified 70-mer oligonucleotide probe performance on glass-slide microarrays." Genome Biol. 2003, 4(1): R5. Epub 2003 Jan 6.

FAQ ID -854
What is the composition of Buffer PB?
Buffer PB contains a high concentration of guanidine hydrochloride and isopropanol. The exact composition of Buffer PB is confidential. However, this buffer can be purchased separately: Buffer PB.
FAQ ID -2791
Do you have a protocol for obtaining > 20 µg plasmid DNA using the QIAprep Spin Miniprep Kit?
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
Do you have a protocol for the isolation of plasmid DNA from Agrobacterium?
FAQ ID -898
Can the QIAprep Spin Miniprep Kit be used for isolating plasmid DNA from mammalian cells?

Yes, it is possible to isolate plasmid DNA from mammalian cells using the QIAprep Spin Miniprep kit . The article in QIAGEN News 1995 No. 2, page 11, Isolation of plasmid DNA from mammalian cells using QIAprep kit, describes a procedure that requires only 30 minutes compared to the time-consuming and labor-intensive Hirt method. 

 

FAQ ID -795
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
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 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
What is the composition of Buffer EB?

The composition of Buffer EB is:

  • 10 mM Tris-Cl, pH 8.5

Buffer EB is the elution buffer used in the QIAquick PCR, Gel Extraction, Nucleotide Removal Kits, and MinElute Kits for DNA cleanup, and the QIAprep Miniprep Kits for small-scale plasmid purification. The purified DNA can also be eluted in TE (10 mM Tris-Cl, 1 mM EDTA, pH 8.0), but the EDTA may inhibit subsequent enzymatic reactions.

FAQ ID -199
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