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

Product FAQ

Answer

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

Was this answer helpful?

Yes
No
b5f40a66ad37749bdbee9701a9021d6e_FAQ

What is the specific activity of your DNase I?

Product FAQ

Answer

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

Was this answer helpful?

Yes
No
c091aaec1575ca85e39fa329490a1de9_FAQ

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

Product FAQ

Answer

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

Was this answer helpful?

Yes
No
b59f0601e0994fa144e3c8503baccf90_FAQ

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

Product FAQ

Answer

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.lifetechnologies.com with the Cat. No.18068-015. The manual will be one of the links on the product page.

Answer Id: E2952

Was this answer helpful?

Yes
No
f5b681a8d2fa8d2d7036a44a553ae21c_FAQ

How can RNA be treated to remove residual DNA?

Product FAQ

Answer

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

Was this answer helpful?

Yes
No
1155a60f7f66fbf08472daca55adfa6d_FAQ

Are your DNase I products RNase-free?

Product FAQ

Answer

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

Was this answer helpful?

Yes
No
808eac3cdf97d0f48a0bf43c48ce2dbd_FAQ

How can I inactivate DNase I?

Product FAQ

Answer

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

Was this answer helpful?

Yes
No
631af69c0bfcae0600b424e77e6e2cc6_FAQ

What is the expected half life of AmpliTaq® DNA Polymerase at 95 degrees C?

Product FAQ

Answer

The half-life of AmpliTaq® DNA Polymerase at 95 degrees C is 40 min. During PCR, the sample is only incubated at the programmed temperature for approximately 20 seconds. Therefore, the cycling half-life of AmpliTaq Gold at 95 degrees C is approximately 100 cycles.

Example: AmpliTaq® DNA Polymerase experiences about 20 seconds at 95 degrees C per PCR cycle. The t1/2 is at least 33 minutes; (35-40 min). Therefore, 33 min/20 sec/cycle = 100 cycles. 100 PCR cycles reduces enzyme activity by 50%.

Answer Id: E1139

Was this answer helpful?

Yes
No
11f0398279d14cfe21e3a1adc1654dae_FAQ

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

Product FAQ

Answer

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

Was this answer helpful?

Yes
No
46e88c55f740097b61c797da6cd46a34_FAQ

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?

Product FAQ

Answer

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

Answer Id: E1235

Was this answer helpful?

Yes
No
2ab0b855b6b0a1e5060074738db37d8c_FAQ

Will adding EDTA prior to heat-inactivation of DNase I inhibit reverse transcription with SuperScript® polymerase?

Product FAQ

Answer

No. After the addition of EDTA, there is an approximately 1:1 molar ratio of Mg++ : EDTA. EDTA chelates Mg++ 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 Mg++. 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.

Answer Id: E4091

Was this answer helpful?

Yes
No
0408dc92e1a1f3b3f6250b5ba475c822_FAQ

Does Life Technologies™ offer a protease-free DNase?

Product FAQ

Answer

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.

Answer Id: E4144

Was this answer helpful?

Yes
No
aedc47c0863a8e674ef66b0b809f14b2_FAQ

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

Product FAQ

Answer

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

Was this answer helpful?

Yes
No
218950219c4ca36b372c087a2488c5c6_FAQ

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

Product FAQ

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

Was this answer helpful?

Yes
No
083f43b7b2f217950f23eff08c7b2e8f_FAQ

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

Product FAQ

Answer

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

Was this answer helpful?

Yes
No
d9d5efe35af3069c564399869c284faa_FAQ