How can RNA be treated to remove residual DNA?

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RNAse-free DNase treatment of the RNA can reduce DNA to undetectable levels. We recommend using our DNase 1, Amplification Grade (Cat. No. 18068015).

Answer Id: E7318

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I don't want to stock twenty different resins for each amino acid. Can I buy a peptide synthesis resin without an amino acid and attach it myself?

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Yes. Resins for making peptide amides have no amino acid, nor do they need one. They have an Fmoc-protected amine.

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Does Life Technologies™ offer a protease-free DNase?

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We do not test for protease activity as part of our QC but there is PMSF (a protease inhibitor) in the storage buffer. Furthermore, in the preparation of DNase I, we uses a soybean trypsin inhibitor column to remove proteases.

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How should I treat my RNA sample prior to RT-PCR to ensure that I have no DNA contamination?

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DNase I treatment is optional, and one has to consider individual experimental design.

Potential disadvantage of omitting the DNase I step: you may get amplification from genomic DNA. If you omit this step, you will need to include a no RT control and design primers that will not amplify genomic DNA, like those spanning two different exons or exon-exon junctions.

Potential benefit of omitting the DNAse I Step:
saves time; consumes less reagent, saves pipetting steps, and reduces RNA loss (important for precious samples).

Protocol for DNAse I treatment:
Combine 1 μg total RNA, 1 μL 10X DNAse I buffer (200 mM Tris-HCl (pH 8.4), 500 mM KCl, 20 mM MgCl2), 1 μL Amplification Grade DNAse I (1 unit/μL), and DEPC-treated water to 10 μL. Incubate for 15 min at room temperature. Inactivate by adding 1 μL of 25 mM EDTA and heat for 10 min at 65 degrees C.
Note: 1 U of DNAse I should be enough to treat up to ~10 μg of RNA.

To locate the manual for Amplification Grade DNAse I, search www.thermofisher.com with the Cat. No.18068-015. The manual will be one of the links on the product page.

Answer Id: E2952

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I have heard I should use HATU in peptide synthesis, but it is so expensive. Do I really need it all the time?

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No, for simple peptides and easy couplings, it may not be necessary. However, if you use a less expensive activator and the peptide needs to be made again, you may have lost any cost benefit.

Answer Id: E1233

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What is the difference between DNase I and Amplification Grade DNase I?

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The Amplification Grade DNase I (Cat. No. 18068-015) is subjected to an extra final HPLC purification step to remove traces of RNases. The Amplification Grade DNase I is supplied as 1 unit/μL and comes with 10X buffer (200 mM Tris-HCL pH 8.4, 20 mM MgCl2, 500 mM KCl) and a vial of 25mM EDTA.

In RT-PCR, a large excess of Amplification Grade DNase I could be used to digest an RNA template without degradation of the RNA (in-house data). Use Amplification Grade DNase I to remove genomic DNA carryover in RNA samples prior to RT-PCR.

The regular DNase I is supplied at 5-15 mg/mL (50-375 U/μL) and does not come with its own buffer.

Answer Id: E2946

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How can I inactivate DNase I?

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Add of 1 µl of 25 mM EDTA solution to the reaction mixture in 10 ul reaction with 1 unit DNase I, Amplification Grade (or 1:1 molar ratio of Mg++:EDTA) to chelate the Mg++ ions in the DNase I buffer. Heat for 10 min at 65°C.

Please Note: It is vital that the EDTA be added to at least 2 mM prior to heat-inactivation to avoid Mg dependant RNA hydrolysis.

DNA-free and Turbo-free versions of DNase I can be inactivated with included DNase Inactivation Reagent.

Answer Id: E2945

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What is the specific activity of your DNase I?

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Specific activities vary by lot. Our DNase I must have a specific activity greater than 10,000 units/mg to pass our quality standards. Average specific activities vary between 10,000-25,000 units/mg.

Answer Id: E2944

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What is the specific activity of your DNase I products, SKU # 18047019 and 18068015?

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Specific activities vary by lot. Our DNase I must have a specific activity greater than 10,000 units/mg to pass our quality standards.

Answer Id: E2943

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Are your DNase I products RNase-free?

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Most of our DNase I products are guaranteed free of RNase activity. However, please note that product 18047-019 is not tested for RNAse and is recommended primarily for protein applications. The other products are suitable for removing DNA from both RNA and protein preparations, for nick translating DNA, and for generating random fragments of DNA. For more demanding RT-PCR applications, we recommend using DNAse I, Amplification Grade.

Answer Id: E2942

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How can I eliminate DNA contamination from my RNA prior to RT-PCR?

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We recommend using Amplification-Grade DNase I, Cat. No. 18068-015, or an equivalent product to eliminate DNA contamination. Combine 1 μg total RNA, 1 μL 10X DNAse I buffer (200 mM Tris-HCl (pH 8.4), 500 mM KCl, 20 mM MgCl2), 1 μL DNAse I, Amplification Grade, 1 unit/μL, and DEPC-treated water to 10 μL. Incubate for 15 min at room temperature. Inactivate by adding 1 μL of 25 mM EDTA and heat for 10 min at 65 degrees C. Note: 1 unit of DNAse I should be enough to treat up to ~10 μg of RNA. The detailed protocol can be found in the product manual. Simply search the Catalog Number on our website to find a copy on the product detail page.

Answer Id: E2916

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I’m getting unexpected bands after electrophoretic analysis of my amplified RT-PCR products. Can you please offer some suggestions?

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Answer

Please see the following causes and suggestions:
Contamination by genomic DNA or an unexpected splice variant - Pretreat RNA with DNase I, amplification grade (Cat. No 18068015).
Design primers that anneal to sequences in exons on both sides of an intron or at the exon/exon boundary of the mRNA to differentiate between amplified cDNA and potential contaminating genomic DNA.
To test if products were derived from DNA, perform a minus RT control.
Nonspecific annealing of primers - Vary the PCR annealing conditions.
Use a hot-start PCR polymerase.
Optimize magnesium concentration for each template and primer combination.
Primers formed dimers - Design primers without complementary sequences at the 3’ ends.

Answer Id: E7331

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How can I remove genomic DNA contamination from my sample prior to performing RT-PCR?

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If amplification products are generated in the control tube/well that contains no reverse transcriptase (i.e., the no-RT control), it may be necessary to eliminate residual genomic DNA from the RNA sample. Use the following protocol to remove genomic DNA from the total RNA preparation.Random primers are the best choice for degraded RNA, RNA with heavy secondary structure, non-polyadenylated RNA, or prokaryotic RNA. It is recommended only for two-step RT-PCR, and typically gives the highest yields, although the cDNA may not necessarily be full length. Oligo(dT) primers are good to use when trying to recover full-length cDNA from 2-step RT-PCR. The reaction is influenced by secondary structure and RNA quality. Gene specific primers should be used for very specific, mainly one-step RT-PCR reactions. Random primers are the best choice for degraded RNA, RNA with heavy secondary structure, non-polyadenylated RNA, or prokaryotic RNA. It is recommended only for two-step RT-PCR, and typically gives the highest yields, although the cDNA may not necessarily be full length. Oligo(dT) primers are good to use when trying to recover full-length cDNA from 2-step RT-PCR. The reaction is influenced by secondary structure and RNA quality. Gene specific primers should be used for very specific, mainly one-step RT-PCR reactions.

Add the following to an autoclaved 0.5 mL microcentrifuge tube on ice:
1.Total RNA, ideally, less than or equal to 1 μg. (See Note 1 below.)
2.1.0 μL of 10X DNase buffer (200 mM Tris, pH 8.3, 500 mM KCl, 20 mM MgCl2).
3.0.1 U-3.0 U of DNase I (RNase-free, Cat. No. 18047019) or 1.0 U Dnase I, Amplification Grade (Cat. No. 18068015. (See Note 2 below.)
4.Bring volume up to 10 μL with DEPC-treated water.
5.Incubate at room temperature for 15 min. (See Note 3 below.)
6.Terminate the reaction by adding 1 μL 25 mM EDTA and heat 10 min at 65 degrees C. (See Note 4 below.)
7.Place on ice for 1 minute.
8.Collect by brief centrifugation. This mixture can be used directly for reverse transcription.

Please note the following:
1.To work with higher quantities of RNA, scale up the entire reaction linearly. Do not exceed 2 μg RNA in the 10 μL reaction. More RNA will increase the viscosity of the solution and prevent the DNAse I from diffusing and finding the DNA.
2.DNAse I, Amplification Grade has been extensively purified to remove trace ribonuclease activities commonly associated with other "RNAse-free" enzyme preparations and does not require the addition of placental RNAse inhibitor.
3.It is important not to exceed the 15 minute incubation time or the room temperature incubation. Higher temperatures and longer times could lead to Mg2+-dependent hydrolysis of the RNA.
4.This procedure requires careful pipetting of all solutions so that the concentration of divalent metal cation (Mg2+) is controlled.
5.Because the DNAse I must be heated to 65 degrees C to inactivate the enzyme, the concentration of free divalent metal ions must be low enough (less than 1 mM) after addition of the EDTA to prevent chemical hydrolysis of the RNA. See references below.
After the addition of EDTA, there is an approximately 1:1 molar ratio of Mg2+ :EDTA. EDTA chelates Mg2+ molecules on a 1:1 molar basis. Therefore, this RNA can be directly used in a reverse transcription reaction. First-strand reverse transcription buffers typically result in a final concentration of 2.5 mM Mg2+. If the reverse transcription buffer does not contain MgCl2, add it to the reaction at a final concentration of 2.5 mM. This results in a net final concentration of approximately 2.25 to 2.5 mM MgCl2.

References on RNA hydrolysis:
Molekulyarnaya Biologiya (1987) 21:1235-1241.
References on the mechanism of hydrolysis by other cations:
Eichorn GL and Butzov JY (1965) Biopolymers 3:79.
Butzov JY and Eichorn GL (1965) Biopolymers 3:95.
Farkas WR (1968) Biochim Biophys Acta 155:401.
The authors of the first paper express the opinion that the mechanism of the nonspecific hydrolysis by cations which proceeds through 2’,3’ cyclic phosphate formation is similar to that of specific hydrolysis such as RNA splicing.

Answer Id: E7322

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