I am trying to use an optional DNA treatment from Amresco company, check attached documents for product details.
I am not quite familiar with DNAse units. So I am confused.
The product I bought says size is 100KU. The certificate of analysis says Activity (Kunitz, Protein) @25C is 1800 units/mg.
The product usage document says I need to prepare a working stock of 0.25 Kunitz unit/μL DNase I in 20 mM Tris, pH 8.3, 2 mM MgCl2. A 100 μL aliquot is required for sample.
Can someone help me explain what a Kunitz unit is, and how to make these calculations. I was assuming 100 KU means kilo units or something like that when I ordered.
Hello Sridhar,
look here: http://www.qiagen.com/products/accessories/rnase-freednaseset.aspx?ShowInfo=1
"One Kunitz unit is defined as the amount of DNase I that causes an increase in A260 of 0.001 per minute per milliliter at 25°C, pH 5.0, with highly polymerized DNA as the substrate"
1. Kunitz, M. (1950) Crystalline desoxyribonuclease. I. Isolation and general properties, spectrophotometric method for the measurement of desoxyribonuclease activity. II. Digestion of thymus nucleic acid (desoxyribonucleic acid): the kinetics of the reaction. J. Gen. Physiol. 33, 349 and 363.
Hello Sridhar,
look here: http://www.qiagen.com/products/accessories/rnase-freednaseset.aspx?ShowInfo=1
"One Kunitz unit is defined as the amount of DNase I that causes an increase in A260 of 0.001 per minute per milliliter at 25°C, pH 5.0, with highly polymerized DNA as the substrate"
1. Kunitz, M. (1950) Crystalline desoxyribonuclease. I. Isolation and general properties, spectrophotometric method for the measurement of desoxyribonuclease activity. II. Digestion of thymus nucleic acid (desoxyribonucleic acid): the kinetics of the reaction. J. Gen. Physiol. 33, 349 and 363.
If you actually have purchased 100 Kunitz units, then you would be adding 400 ul of 20 mM Tris, pH 8.3, 2 mM MgCl2 to obtain a 0.25 U/ul stock. How much of this stock are you supposed to be adding to your DNAse reaction?
An important point is whether the DNAse that you bought is certified RNAse free. (And whether you are certain that the reagents you have to resuspend it are also RNase free!) If you are uncertain about that, you might consider the Promega product.
http://www.promega.com/resources/protocols/product-information-sheets/g/rq1-rnasefree-dnase-protocol/
This product has the advantage that the units are defined in terms of how much you need to treat an RNA sample of a certain size (no mention of the enzymologist's Kunitz unit!) RNase free buffer is provided for use with this DNAse, but you should strongly consider adding an RNAse inhibitor to the reaction also. Depending on the source of your RNA, there may be substantial residual RNAse present and this will happily chew up your RNA during the incubation required for the DNAse to work! Promega also sells RNasin which can be added to the DNAse reaction. There are other suppliers of such RNase inhibitors and you should consider adding one to your DNAse reaction, with whatever product you decide to use.
I have a question regarding this Kunitz units conversion. I have 1500kunitz units of lyophilized powder from Qiagen and I want to incubate my tissue sections which I will be using for my TUNEL (I mean I want to treat them with DNAse to serve as a positive control). So the TUNEL protocol says I need 3000U/ml of DNAse 1 in 50mM Tis -HCL, ph=7.5, 1mg/mL BSA. I am still not clear how to do this conversion????
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