What is the specific activity of your DNase I products, SKU # 18047019 and 18068015?
What is the expected half life of AmpliTaq® DNA Polymerase at 95 degrees C?
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: 1139
I have heard I should use HATU in peptide synthesis, but it is so expensive. Do I really need it all the time?
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?
Are your DNase I products RNase-free?
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: 2942
How can I inactivate DNase I?
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: 2945
Will adding EDTA prior to heat-inactivation of DNase I inhibit reverse transcription with SuperScript® polymerase?
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: 4091
Does Life Technologies™ offer a protease-free DNase?
What is the specific activity of your DNase I?
What is the difference between DNase I and Amplification Grade DNase I?
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: 2946
How should I treat my RNA sample prior to RT-PCR to ensure that I have no DNA contamination?
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: 2952
How can I eliminate DNA contamination from my RNA prior to RT-PCR?
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: 2916