What is RNase-free DNase?
Thermo Scientific DNase I, RNase-free is an endonuclease that digests single- and double-stranded DNA. It hydrolyzes phosphodiester bonds producing mono- and oligodeoxyribonucleotides with 5′-phosphate and 3′-OH groups. The enzyme activity is strictly dependent on Ca2+ and is activated by Mg2+ or Mn2+ ions.
How do you use DNase 1?
A Typical DNase I Reaction Protocol (M0303)
- Set up the following reaction on ice: COMPONENTS. 100 μl REACTION. RNA. ~ 10 μg RNA. DNase I Reaction Buffer (10X) 10 μl (1X) DNAse I (RNase-free)
- Incubate at 37°C for 10 minutes.
- Add 1 µl of 0.5 M EDTA (to a final concentration of 5 mM).
- Heat inactivate at 75°C for 10 minutes.
Will DNase I digest RNA?
For removal of genomic DNA from RNA samples, a DNase I treatment is recommended. The DNAse I enzyme is classified as an endonuclease which is able to digest single and double-stranded DNA into single bases or oligonucleotides.
What does DNase treatment do?
Getting Rid of Contaminating DNA and the DNase Used to Destroy it. Because virtually all RNA samples have trace amounts of contaminating DNA, most protocols specify DNase treatment for RT-PCR applications. DNase I treatment is clearly the best way to rid an RNA sample of contaminating DNA.
How long does DNase treatment take?
Heat inactivation: Probably the most common method of DNase inactivation is heat treatment, typically for 5 minutes at 75°C.
How long is DNase I good for?
3. For long-term storage of DNase I, remove the stock solution from the glass vial and divide it into single-use aliquots. Aliquots can be stored at –15 to –25°C for up to 9 months. Thawed aliquots can be stored at 2–8°C for up to 6 weeks.
How can you remove RNA from DNA sample without RNase?
If you really think RNA to be interfering with your reactions, you can re-purify your extracts with alkaline-Phenol-chloroform method. Alternatively you simply run your extracts on an 0.8% agarose gel, slice the gDNA band from the gel and purify with a gel DNA extraction kit.
What is rnase and DNase?
The function of nucleases (DNases and RNases) includes the enzymatic breakdown of DNA and RNA and is necessary for numerous research applications. For example, the purification of proteins and specific nucleic acids often requires the digestion of DNA, RNA or both.
Is DNase treatment necessary?
Because con- struction of such primers is complicat- ed, the most commonly used method to avoid signals from the genomic DNA is to treat the tissue with DNase (7). Since this treatment is necessary to eliminate all DNA, it is a very crucial part of the in situ RT-PCR protocol.
What is QIAzol?
QIAzol is optimized for lysis of fatty tissues, allowing recovery of high-quality RNA from adipose tissue, especially when used in combination with RNeasy silica membrane technology (see figure ” High-quality RNA”).
How do you extract a small RNA?
Small RNA extraction
- Place 0.1 g of pulverized frozen tissue in a 1.5 ml microcentrifuge tube and add 500 μl of LiCl extraction buffer and 500 μl of phenol pH 8.0.
- Shake or mix well using a vortex for 1 min.
- Incubate tubes for 5 min at 60°C.
- Centrifuge for 10 min in a microcentrifuge at max speed at 4°C.
How quickly does DNase work?
One unit of DNase I, Amplification Grade, completely digests 1 ug of plasmid DNA to oligonucleotides in 10 minutes at 37 °C. As long as your total DNA content is less than 1ug you should be ok with just 1uL. You can also incubate for longer than 10 minutes to make sure the digest is complete.