QIAquick PCR Purification Kit for PCR Cleanup

最大10 µgのPCR産物100 bp ～ 10 kbの精製に

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QIAquick PCR Purification Kit (50)

Cat. No. / ID: 28104

For purification of 50 PCR reactions:50 QIAquick Spin Columns, Buffers, Collection Tubes (2 ml)

￥16,000

キットカラム

QIAquick PCR Purification Kit

QIAquick PCR & Gel Cleanup Kit

QIAquick Spin Columns

調製

250

QIAquick PCR Purification 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。

✓ オンライン注文による24時間年中無休の自動処理システム

✓ 知識豊富で専門的な製品&テクニカルサポート

✓ 迅速で信頼性の高い（再）注文

特徴

すぐに使えるDNAの最大95％の回収率
3つの容易なステップで最大10 kbのDNAのクリーンアップ
サンプル操作を便利にするローディングダイ
ゲル抽出かつPCRクリーンアップ用のキット
スピンカラムは別売です

製品詳細

QIAquick PCR Purification Kitは、>100 bpのPCR産物精製のシリカメンブレンスピンカラム、バッファー、およびコレクションチューブで構成されます。最大10 kbのDNAを、シンプルで迅速な結合－洗浄－溶出の操作および30–50 μlの溶出液量で精製します。オプションのpH指示薬で、スピンカラムへのDNA結合の至適pHを容易に確認できます。この操作は、QIAcube Connectで完全自動化できます。

最適な結果を得るには、本品をQIAvac 24 Plusと併用することをお勧めします。

QIAquick PCR Purificationの標準プロトコールは、PIPETMAN M Connectedピペットと組み合わせたTRACKMAN Connectedシステムで実行することもできます。いずれもGilsonの製品です。TRACKMAN Connectedシステムは、QIAquick PCR Purificationプロトコールを通して研究者を導く一方で、Bluetoothで有効化するPIPETMAN M Connectedピペットセッティングを自動的に調整します。プロトコール実行の各ステップは、総合的なランレポートを作成することにより報告を加速化するために記録されます。詳細情報をダウンロード。

パフォーマンス

QIAquick PCR Purificationの手順は、DNAサンプルからプライマー、ヌクレオチド、酵素、ミネラルオイル、塩などの不純物を除去します（「」の図を参照）。小型遠心機または真空マニホールドを使用して、100 bp ～ 10 kbのDNAを精製します。

図参照

PCR後の完全なプライマー除去。

原理

QIAquick Kitsは、高塩濃度バッファーでDNAを結合し、低塩濃度バッファーまたは水で溶出するためのシリカメンブレン技術です。この精製操作は、DNAサンプルからプライマー、ヌクレオチド、酵素、ミネラルオイル、塩、アガロース、臭化エチジウムなどの不純物を除去します（「 PCR後の完全なプライマー除去」の図を参照）。シリカメンブレン技術は、緩い樹脂やスラリーに伴う問題や不都合がありません。特殊な結合バッファーは、特定のアプリケーション向けに最適化され、特定のサイズ範囲内でのDNA分子の選択的吸着を促進します。

Gel loading dye

ローディングダイは、迅速で便利なサンプル処理と分析を可能にします。GelPilot Loading Dyeは、3種類のトラッキングダイ（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）を含み、アガロールゲルのランタイムの最適化を容易にし、より小さなDNAフラグメントの過度な移動を防ぎます（「 GelPilot Loading Dye」の図を参照）。

図参照

PCR後の完全なプライマー除去。

GelPilot Loading Dye。

操作手順

QIAquickシステムは、シンプルな結合－洗浄－溶出の操作です（「 QIAquickおよびMinEluteの操作手順」のフローチャートを参照）。結合バッファーは、PCRサンプルまたは酵素反応液に添加し、混合液をQIAquickスピンカラムにアプライします。結合バッファーはpH指示薬を含んでおり、DNA結合の至適pHを容易に確認できます（「pH Indicator Dye」図を参照）。核酸は、バッファーの高塩濃度の条件でシリカメンブレンに吸着します。不純物は洗い流され、純粋なDNAは少量の低塩濃度バッファーまたは水と共に溶出し、様々なアプリケーションに使用できます。

取り扱い

QIAquickスピンカラムは、2通りの操作が設定されてます。スピンカラムは、小型遠心機またはQIAvac Luer Adapters付きのQIAvac 24 Plusなどのルアーコネクター付きの真空マニホールド上にフィットします。 QIAquick PCR Purification Kitは、その他のQIAGENスピンカラムキットに加えて、QIAcubeで完全自動化すして、生産性の向上、結果の標準化することができます（「スピンカラムの取り扱いオプション A、 B、 C、 D」および「 QIAcube Connect」の図を参照）。

図参照

QIAquickおよびMinEluteの操作手順。

アプリケーション

QIAquickシステムで精製したDNAフラグメントは、シークエンシング、マイクロアレイ解析、ライゲーションと形質転換、制限酵素消化、標識、マイクロインジェクション、PCR、およびin vitro転写など多くのアプリケーションで使用できます。

裏付けデータと数値

QIAquickおよびMinEluteの操作手順。

QIAquickおよびMinEluteシステムは、スピンカラムまたは真空マニホールドでシンプルな結合－洗浄－溶出の手順を用いています。

Specifications

Features	Specifications
Binding capacity	10 µg
Technology	シリカテクノロジー
Sample type: applications	PCRやその他の酵素反応由来のssDNAまたはdsDNA
Fragments removed	＜40mers
Elution volume	＞30 µl
Fragment size	100 bp ～ 10 kb
Processing	手動
Format	チューブ
Type(s) of DNA recovered	ss DNAとdsDNA

リソース

The protocol has been used successfully for Cy3-, Cy5-, and biotin-labeling of cDNA from <50 ng of total RNA or poly A⁺ mRNA.

Download Safety Data Sheets for QIAGEN product components.

FAQ

Larger DNA fragments bind more tightly to the QIAquick columns. It is difficult to predict whether a DNA fragment larger than 10 kb can be efficiently recovered, because this depends on base composition as well as fragment size. If the fragment is only a few kb larger than the 10 kb limit, it can be helpful to heat the elution buffer EB to 60°C and let it incubate on the column for a few minutes before centrifuging. However, please note that it will become less likely to recover your sample the larger the fragment size is. As we cannot guarantee recovery of fragments larger than the maximum cutoff size, we do not recommend to purify such fragments using QIAquick Kits.

The QIAEX II Kit can be used to extract DNA fragments up to 50 kb from agarose or polyacrylamide gels.

FAQ ID -756

Buffer PB of the QIAquick PCR Purification Kit cannot be used to extract DNA from agarose gels. However, Buffer QG of the QIAquick Gel Extraction Kit can be used to remove salt and proteins from enzymatic reactions by adding 3 volumes of Buffer QG and 1 volume of isopropanol to the reaction and proceeding with step 6 of the Gel Extraction Spin Protocol in the QIAquick Spin Handbook. See the QIAquick Spin Handbook for a list of reactions which can be cleaned up with the various QIAquick kits.

FAQ ID -786

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

Yes - all QIAquick Spin Kits contain identical columns, but different binding buffers optimized for each specific application.

FAQ ID -577

Buffer PE did not contain ethanol

Ethanol must be added to Buffer PE (concentrate) before use. Repeat procedure with correctly prepared Buffer PE.

Inappropriate elution buffer

DNA will only be eluted efficiently in the presence of low-salt buffer (e.g., Buffer EB: 10 mM Tris·Cl, pH 8.5) or water. Elution efficiency is strongly dependent on the salt concentration and pH of the elution buffer. Contrary to adsorption, elution is most efficient under basic conditions and low salt concentrations. DNA is eluted with 50 or 30 µl of the provided Buffer EB (10 mM Tris·Cl, pH 8.5), or water. The maximum elution efficiency is achieved between pH 7.0 and 8.5. When using water to elute, make sure that the pH is within this range. In addition, DNA must be stored at –20°C when eluted with water since DNA may degrade in the absence of a buffering agent. Elution with TE (10 mM Tris·Cl, 1 mM EDTA, pH 8.0) is possible, but not recommended because EDTA may inhibit subsequent enzymatic reactions.

Elution buffer incorrectly dispensed

Add elution buffer to the center of the QIAquick membrane to ensure that the buffer completely covers the membrane. This is particularly important when using small elution volumes (30 µl).

FAQ ID -180

Although it is possible to use the QIAquick Nucleotide Removal Kit and QIAquick PCR Purification Kit for RNA purification, the conditions are not optimized for RNA, nor are RNase-free conditions guaranteed. The RNeasy MinElute Cleanup Kit is recommended for clean-up, concentration and desalting of RNAs above 200 bases in length.

FAQ ID -490

CoralLoad dyes supplied in PCR Kits such as, e.g., Taq, HotStarTaq, and TopTaq DNA Polymerase and TopTaq Master Mix do not interfere with most downstream enzymatic applications.

However, for reproducible results, purification of PCR products using the QIAquick or MinElute PCR Purification Kits prior to enzymatic manipulation is recommended.

FAQ ID -1745

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

Yes, please follow the Supplementary Protocol 'Purification of DNA fragments from dye-labeled reactions using the QIAquick PCR Purification Kit' (QQ06).

FAQ ID -947

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

Based on our experience, the QIAquick PCR Purification Kit removes SYBR Green dye efficiently from PCR reactions.

We strongly assume that the QIAquick Gel Extraction Kit will be equally efficient in removing this dye, however, we recommend a 5 min incubation with wash Buffer PE on the QIAquick spin column at step 10 of the QIAquick Gel Extraction Kit Protocol. Efficiency of SYBR Green dye removal has to be validated by the enduser.

FAQ ID -637

No - mineral oil will not affect the clean-up procedure with the QIAquick or MinElute PCR Purification Kit.

FAQ ID -575

Yes. The QIAquick PCR Purification Kit has been used to clean up fragments between 100 bp and 10 kb from a wide range of enzymatic reactions, removing salts, buffers, enzymes, nucleotides, and primers smaller than 40 nucleotides. Reactions that can be cleaned up with the QIAquick PCR Purification Kit include restriction digests, random priming, ligase, kinase, phosphatase, nuclease, nick translation, and cDNA synthesis reactions.
For data and additional information, please see QIAGEN News article Issue No. 5, 1998 "Fast and efficient enzyme removal with QIAquick Spin kits."

FAQ ID -130

DNA fragments purified with the QIAGEN DNA Cleanup Systems, i.e., the QIAquick PCR Purification Kit, the MinElute Reaction Cleanup Kit, the QIAEX II Gel Extraction Kit etc. may float out of the loading wells of agarose gels due to residual ethanol carried over from the wash step with Buffer PE (despite the addtition of glycerol-containing loading buffer).

Use either of the following options to remove residual ethanol from the eluate:

re-purify the sample using a QIAquick-, or MinElute column, or QIAEX II resin
incubate the eluate at 56°C for 10 min to evaporate the ethanol
dry down the sample in a vacuum centrifuge, and resuspend the pellet in a small volume of sterile water

FAQ ID -205

Yes, bisulfite containing methylation reactions can be cleaned up with our silica-based cleanup products, such as QIAquick and QIAEX II. Please see Goyon et al. (1994), 'Perpetuation of cytosine methylation in Ascobolus immersus implies a novel type of maintenance methylase', published in J Mol Biol. 1994 Jul 1;240(1):42-51, for a reference.

FAQ ID -519

As a rule of thumb, single-stranded DNA binds to silica with approximately half the affinity of a double-stranded DNA fragment of the same length under the buffer conditions used in the QIAquick and MinElute Kits. Even though no systematic experimental data exists, we expect that recovery of ssDNA fragments of approximately 200 nucleotides and below will not be very efficient after cleanup using the QIAquick PCR Purification Kit or MinElute PCR Purification Kit. By comparison, it should be possible to purify fragments longer than 140 nucleotides using the QIAquick Gel Extraction Kit.

Note that recovery of single strand DNA is influenced to some degree also by factors such as base composition and secondary structure. It has to be determined empirically by the researcher if cleanup of single-stranded DNA with QIAquick columns yields satisfactory results.

FAQ ID -759

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

Occasionally, DNA fragments eluted from the silica matrix of QIAquick, MinElute or QIAEX II Kits will contain denatured single-stranded DNA (ssDNA), appearing as a smaller band on an analytical gel. Under certain conditions, chaotropic agents (present in all silica-based DNA purification methods) can denature DNA fragments. This is a rare event that may be influenced by sequence characteristics such as the presence of inverted repeats or A–T-rich stretches.

Because salt and buffering agents promote renaturation of DNA strands, the following tips are recommended:

use the eluted DNA to prepare your downstream enzymatic reaction, but omit the enzyme. Incubate the reaction mix at 95°C for 2 minutes to reanneal the ssDNA, and allow the tube to cool slowly to room temperature before adding the enzyme and proceeding
alternatively, the DNA can be eluted from the silica-gel membrane or resin in 10 mM Tris buffer containing 10 mM NaCl. However, the salt concentration of the eluate must then be taken into consideration in downstream applications.

FAQ ID -148

The QIAquick Spin Columns (100) (cat. no. 28115) in the QIAquick PCR Purification, Gel Extraction, Nucleotide Removal and PCR & Gel Cleanup kits are also sold separately from the kits.

The MinElute columns in the MinElute PCR Purification, Gel Extraction and Reaction Cleanup kits are not sold separately.

We always provide extra buffers in our kits so you can scale up reactions, add extra washes or allow for spillage.

FAQ ID -2460