I recently purchased DNase I powder (Type 1A, from Bovine Pancreas, 2000 Kunitz U/mg, 80% protein; Product by SRL, Cat. No. 61824) to eliminate genomic DNA contamination from RNA samples prior to cDNA synthesis. However, the product came without a detailed protocol or accessory materials (Only the 'DNase 1' powder vial), and there is no clear guidance available on the manufacturer's website.
Product Details:
- Name: Deoxyribonuclease I (DNase I) – Type 1A ex. Bovine Pancreas for Molecular Biology
- Activity: 2000 Kunitz U/mg, 80% protein
- Supplier: SRL
- Net content: 10k Units
- Product Page: https://www.srlchem.com/product/details/12/61824/deoxyribonuclease-i-dnase-i---type-1a-ex-bovine-pancreas-for-mb-2000kunitz-umg-80-protein
Thanks in advance!
The standard buffer for doing DNase I digestion is 10 mM Tris-HCl (pH 7.5 at 25°C), 2.5 mM MgCl2, 0.1 mM CaCl2. You can make a 10X stock of for use (the above are the 1X final concentrations). While I would probably dissolve your enzyme in 10mM Tris around pH 7.5 but I don't think there is any problem if you dissolved it in reaction buffer either.
Michael J. Benedik I was facing the same problem as mentioned by Md Shamsudduha . When I went through some documents available online, 0.5mM CaCl2 as final concentration is mentioned
https://assets.thermofisher.com/TFS-Assets/LSG/manuals/4393898B.pdf
Can DNAse 1 be dissolved in water as well? And, after EDTA treatment during inactivation, is it always necessary to re extract the RNA before going to cDNA conversion?
A bit of extra CaCl2 or MgCl2 is not going to hurt anything. You can dissolve DNAse I in water, I just tend to prefer to have some buffer present because water is so variable in terms of its pH.
Adding EDTA does not kill the enzyme, it just stops activity by chelating the needed Mg and Ca ions. However if you add Mg back in subsequent steps then the enzyme will be activate again and digest your newly synthesized cDNA. So I think you do need to re-extract the RNA.
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