DNase, Double-Strand Specific, Heat-Labile

Transcription

1 DNase, Double-Strand Specific, Heat-Labile Code Description Size 1B U DNase Double-Strand Specific, Heat-Labile 1000 units (2 U/µL) 1B U DNase Double-Strand Specific, Heat-Labile 250 units (2 U/µL) General Information VWR Life Science AMRESCO s DNase, Double-Strand Specific, Heat-Labile is a recombinant endonuclease that cleaves phosphodiester bonds in DNA to yield 2 8 bp oligonucleotides with 5 -phosphate and 3 -hydroxyl termini. The high specific activity of this enzyme toward dsdna can be inactivated by heating at 55 C, conveniently eliminating the need for its physical or chemical removal before downstream processing, even in the presence of RNA and ssdna, such as primers and probes. DNase, Double-Strand Specific, Heat-Labile is ideal for removal of genomic DNA in RNA preps and for removal of DNA carryover contamination in PCR mixes, before addition of template. High specificity for dsdna, while leaving ssdna and RNA intact Heat inactivates completely and irreversibly at 55 C Ideal for genomic DNA removal in RNA preps Removes contaminating dsdna from PCR master mixes Storage/Stability Store frozen (-20 0 C). Stable two years when stored frozen (-20 0 C) and up to six months when stored cold (2 8 C). The enzyme tolerates multiple freeze-thaw cycles. Product Use Limitations For research use only. Not for therapeutic or diagnostic use.

2 Protocol/Procedure: Genomic DNA Removal from RNA Preps Decontaminate RNA preps with minimal influence on final RNA concentration, integrity or quality. 1. Elute or re-suspend RNA in nuclease free water after isolation. 2. Add 0.1 Unit of DNase, Double-Strand Specific, Heat-Labile per µl RNA sample. 3. Add MgCl 2 to a final concentration of 3 mm to DNase activity. 4. Add DTT to a final concentration of 1 mm to ensure complete inactivation of the DNase upon heating. Component Volume (µl) Final Concentration RNA 50 DNase, Double-Strand Specific, Heat-Labile 5 50 mm MgCl mm 100 mm DTT mm Nuclease-free Water 0.8 Total Volume Incubate the sample for 15 minutes at room temperature to remove contaminating DNA. 6. Heat inactivate the DNase by incubating 10 minutes at 55 C. The DNA-free RNA from this protocol is compatible with downstream applications, including one-step and two-step RT-PCR.

3 Decontamination of PCR Master Mixes 1. Prepare a PCR master mix, excluding the template DNA. Ideally, the master mix should include 3mM Mg 2+ and 50 mm KCl. 2. Add 1 unit of DNase, Double-Strand Specific, Heat-Labile per 25 µl PCR reaction. Component Volume (µl) Final Concentration PCR Buffer ( MgCl 2), 10X 5.0 1X MgCl 2, 25 mm mm dntp Mix, 10 mm mm each Forward Primer, 10 µm µm Reverse Primer, 10 µm µm Nuclease-free Water 25.5 Taq DNA Polymerase 5 U/µL U DNase, Double-Strand Specific, Heat-Labile U Total Master Mix Volume 45.0 As needed to bring final volume to 50 µl Template DNA *To be added in step ng (5 µl) 3. Incubate the sample for 10 minutes at 37 C to remove contaminating DNA. 4. Heat inactivate the DNase by incubating 10 minutes at 55 C 5. Cool, then add template DNA to PCR master mix. 6. Proceed with PCR reaction.

4 Frequently Asked Questions What are the optimal reaction conditions for DNase, Double-Strand Specific, Heat-Labile? Reaction Temperature Salt Concentration ph 7.5 Units Per Reaction Minimal Substrate Length End-product Length Optimal Conditions C activity range; 40 C optimum 2.5 mm Mg 2+ (Activity increases with increasing concentration up to 10 mm Mg 2+.) DNase is less active in buffers containing > 10 mm KCl and NaCl. Avoid concentrations above 50 mm. (NH4)2SO4 inhibits activity. The general recommendation for a 50 µl reaction is 2 units. Increasing the number of units may be beneficial in some reactions if high ph, salt or other inhibiting factor results in decreased activity. 9 10bp dsdna Cleavage products are about 5 bp ±3 bp How is DNase, Double-Strand Specific, Heat-Labile different than standard dsdnase? DNase, Double-Strand Specific, Heat-Labile is inactivated after 5 minutes at 55 C, while dsdnase requires 15 minutes incubation at 65 C for inactivation. Does DNase, Double-Strand Specific, Heat-Labile cleave single-stranded DNA? No, it does not cleave ssdna, only dsdna. DNase, Double-Strand Specific, Heat-Labile exhibits a minimum of 5,000-fold higher activity for dsdna than for ssdna. However, if ssdna forms double-stranded regions internally or by binding other ssdna during treatment, it will be targeted by the enzyme. Therefore, complex and longer ssdna (e.g. cdna) may be partially degraded. What advantage does DNase, Double-Strand Specific, Heat-Labile offer compared to nonthermolabile DNases? VWR Life Science AMRESCO s Heat-Labile DNase is completely and irreversibly inactivated at a sufficiently low temperature to enable treatment in the presence of RNA or ssdna and probes. This eliminates the need to physically or chemically remove the enzyme before downstream processing.. Does DNase, Double-Strand Specific, Heat-Labile digest DNA within DNA-RNA hybrids? The DNase may digest the DNA strand in DNA-RNA hybrids under the right conditions at a level of 20% compared to the activity toward dsdna. In RT-PCR reactions, the

5 DNase is inactive if the RT incubation step is at 50 C or above. If cdna is synthesized at temperatures lower than 40 C, it may be partially degraded (Refer to question 2). Is DNase, Double-Strand Specific, Heat-Labile suitable for genomic DNA removal in RNA preps? Yes, it is especially recommended for gdna removal in RNA samples because of its low inactivation temperature. Follow the procedure carefully in regard to the MgCl 2 concentration to avoid autocatalysis of RNA in the cleavage mixture. How is DNase, Double-Strand Specific, Heat-Labile useful for decontamination of PCR carry-over? DNase, Double-Strand Specific, Heat-Labile will remove any contaminating dsdna from PCR master mixes, including carry-over amplicons. It is recommended that PCR master mixes, minus the template DNA, be assembled and treated, then heat inactivated before addition of template. PCR is then performed per standard protocols. What is the molecular weight of DNase, Double-Strand Specific, Heat-Labile? Is it a tagged protein? The molecular weight of the enzyme is 42 kda and it is not a tagged protein. How stable is DNase, Double-Strand Specific, Heat-Labile? DNase, Double-Strand Specific, Heat-Labile is stable for at least 2 years when stored frozen and up to 6 months when stored cold. The enzyme can withstand 5 7 days at room temperature without significant loss of activity, but is very unstable if kept at temperatures above 50 C. No significant effect on enzyme activity has been observed upon five freeze-thaw cycles. Does DNase, Double-Strand Specific, Heat-Labile contain BSA? No, there is no BSA in this product.

6 For Technical Support Toll Free: (USA & Canada) Fax: (440) AMRESCO, LLC A VWR Company Corporate Headquarters Fountain Parkway Solon, Ohio USA Tel: 440/ Fax: 440/ DNase, Double Strand Specific Heat Labile ZY0624 Rev. 1 12/2015 Copyright 2010 by AMRESCO, LLC All Rights Reserved. AMRESCO is a registered trademark of AMRESCO, LLC