RNase-Free DNase Set

RNA精製中のDNase処理用

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RNase-Free DNase Set (50)

Cat. No. / ID: 79254

1500 Kunitz units RNase-free DNase I, RNase-free Buffer RDD, and RNase-free water for 50 RNA minipreps

€124.00

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Preparations

250

数量

RNase-Free DNase Set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。

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特徴

Compatible with RNeasy procedures and the QIAamp RNA Blood Mini Kit
Efficient on-column digestion of DNA during RNA purification

製品詳細

QIAGEN RNase-Free DNase Set はRNaseフリーの保証付きで品質管理され RNeasy 操作およびQIAamp RNA Blood Mini 操作との併用に至適化されています。シリカメンブレン、スピンカラムテクノロジーではDNase 処理なしでも効率的にDNAを除去できるので、これらのキットを用いたRNA 精製では通常DNase 処理は必要ありません。しかし非常に微量なDNA にも敏感な、ある種のRNA アプリケーションの際には完全なDNA 除去が必要な場合もあります。セットに含まれているBuffer RDD はカラム上のDNase 分解（20～30℃で15分間）用に至適化されていますが、溶解中での効果的なDNase 分解にも適しています。RNase-Free DNase Set はRNeasy Kit およびQIAamp RNA Blood Mini Kit を用いた細胞および組織からのRNA 精製の際に、カラム上でのDNase 除去を効率的に行ないます。DNase は続いて行なう洗浄ステップで、効率的に除去されます。RNeasy 96 Kitを使用する際のDNase 処理に関しましては、別途至適化されたプロトコールが必要になります。弊社テクニカルサポートにお問い合わせください。QIAGEN RNase-Free DNase Set は安定な凍結乾燥酵素としてお届けします。RNase-Free DNase Set は1500 Kunitz units で提供されます。高分子DNA を基質として、25℃、pH 5.0 において反応液1 mlのA₂₆₀ の吸光度を1 分間に0.001 増加させるDNase 活性を1 Kunitz unit とします（Kunitz, M. [1950] Crystalline desoxyribonuclease）。 I. Isolation and general properties, spectrophotometric method for the measurement of desoxyribonuclease activity.II.Digestion of thymus nucleic acid（desoxyribonucleic acid）: the kinetics of the reaction.J. Gen.Physiol.33, 349 and 363）。

パフォーマンス

One Kunitz unit is defined as the amount of DNase I that causes an increase in A₂₆₀ of 0.001 per minute per milliliter at 25°C, pH 5.0, with highly polymerized DNA as the substrate (Kunitz, M. [1950] Crystalline desoxyribonuclease. I. Isolation and general properties, spectrophotometric method for the measurement of desoxyribonuclease activity. II. Digestion of thymus nucleic acid (desoxyribonucleic acid): the kinetics of the reaction. J. Gen. Physiol. 33, 349 and 363).

リソース

For DNase treatment with QIAGEN or PreAnalytiX RNA purification kits

Download Safety Data Sheets for QIAGEN product components.

FAQ

The RNeasy Midi/Maxi Handbook contains a protocol for the use of the RNase-Free DNase Set for on-column DNA digestion on RNeasy midi or maxi spin columns. Incubation times and reagent volumes have been modified from the standard RNeasy Mini procedure. See Appendix E of the RNeasy Midi/Maxi Handbook for the detailed protocol.

FAQ ID -143

Yes, buffer RDD of the RNase-Free DNase Set will still work. Please make sure that the buffer is thawed completely without any precipitates before using it. If precipitates are visible, the buffer should be slightly heated.

-20

Yes. RNeasy MinElute Cleanup Kit can also be used to clean up RNA following DNase digestion, or from crude RNA preparations following organic extraction or alcohol-precipitation.

FAQ ID -429

The exact composition of Buffer RDD is proprietary. Buffer RDD is an important component of the RNase-Free DNase Set, which is used in combination with most RNeasy Kits. The composition and salt concentration of Buffer RDD provides efficient on-column digestion of DNA and also ensures that the RNA remains bound to the column.

FAQ ID -2800

Yes. Even though buffer RDD in the RNase-Free DNase Set is optimized for on-column DNase digestion, the buffer is also well-suited for efficient DNase digestion in solution. Please note that the reaction must be cleaned up after the off-column DNase digest to remove the enzyme and buffer RDD, which will interfere with subsequent RT reactions.

A protocol for in-solution DNase digestion using the RNase-Free DNase Set can be found in Appendix C of the RNeasy MinElute Cleanup Handbook. For subsequent RNA Cleanup, use either the RNeasy MinElute Cleanup Kit, or follow the instructions for RNA Cleanup in the RNeasy Mini Handbook.

FAQ ID -619

RNeasy 96 Kit procedure requires two RNase-Fee DNase sets (cat. no. 79254) for each 96-well plate.

1. To obtain a concentration of 1.8 Kunitz/µl, dissolve each vial of lyophilized DNase in 833 µl of RNase-free water and combine the reconstituted enzyme solution from both vials into one vial.

2. Take 1 ml of this DNase I solution and mix with 7 ml of Buffer RDD* to prepare a DNase I incubation mix immediately before starting the RNeasy 96 protocol. Vortex briefly and keep on ice until use.

3. Use 80 µl of the DNase I incubation mix (from step 2 above) for each well of the RNeasy 96 plate, to perform on-column DNase digestion, according to instructions in the RNeasy 96 Handbook.

*The RDD Buffer Set for RNeasy 96 is available upon request. Please contact QIAGEN Technical Service for information on ordering. Buffer RDD from QIAGEN is optimized for DNase I digestion on the RNeasy membrane.

FAQ ID -3003

Carry out all procedures in a "DNA-free" workspace (see FAQ 2654). Be sure to include any DNase treatment steps in the recommended RNA isolation procedure or treat RNA separately with RNase-free DNase followed by repurification using a spin-column based method. RNeasy Mini Kit can be easily combined with RNase-free DNase. Alternatively, kits like the RNeasy Plus Universal Tissue already include a DNA removal step. Be sure to double both the units of enzyme and the incubation time recommended by the RNase-free DNase manufacturer. To minimize DNA contamination in your RNA preparations, and avoid the need for supplemental DNase treatments, we recommend using the RT2 First Strand Kit, which includes a highly efficient genomic DNA elimination step before reverse transcription.

NOTE: Our Chemistries are not compatible with AMBION's Turbo DNA-Free Kits.

FAQ ID -2662

To ensure efficient gDNA removal when doing an on-column digest using the RNase-Free DNase Set in combination with RNeasy Mini the following factors are crucial:

prevent overloading by adjusting the amount of starting material to no more than the maximum amounts recommended in the RNeasy Mini Handbook
ensure complete disruption and homogenization of the starting material as instructed in the section 'Disruption and homogenization of starting materials' of the handbook
strictly follow the protocol for on-column DNase Digestion in Appendix D of the RNeasy Mini Handbook (you can let wash buffer RW1 incubate on the column for 3-5 minutes before centrifuging to enhance removal of excess gDNA prior to applying the enzyme)

In the rare case that trace amounts of genomic DNA are still detectable in sensitive downstream applications such as e.g., realtime RT-PCR, an in-solution digest using the RNase-Free DNAase set can be performed. Instructions are presented in Appendix C of the RNeasy MinElute Cleanup Handbook.

Alternatively, a second on-column digest can be carried out in future preparations, immediately following the RW1 wash after the first incubation with DNase.

FAQ ID -1087