Kit de extracción de ADN DNA-free™

Add of 1 µL of 25 mM EDTA solution to the reaction mixture in 10 µL reaction with 1 unit DNase I, Amplification Grade (or 1:1 molar ratio of Mg++ ions:EDTA) to chelate the Mg++ ions in the DNase I buffer. Heat for 10 min at 65 degrees C.

Note: It is vital that the EDTA be at least at 2 mM prior to heat-inactivation to avoid Mg-dependent RNA hydrolysis.

DNA-free and Turbo-free versions of DNase I can be inactivated with included DNase Inactivation Reagent.

Most of our DNase I products are guaranteed free of RNase activity. However, please note that Cat. No. 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.

There are two methods for DNase treatment depending on the amount of contaminating DNA and the nucleic acid concentration of the sample.
- Routine DNase treatment: Sample contains ≤200 µg nucleic acid per mL. Use 1 µL rDNase I (2 U) for up to 10 µg of RNA in a 50 µL reaction. These reaction conditions will remove up to 2 µg of genomic DNA from total RNA in a 50 µL reaction volume. For additional details, see ''Perform routine DNase treatment'' on page 3 of the user manual.
- Rigorous DNase treatment: Sample contains >200 µg nucleic acid per mL. Dilute the sample to 2 µg of nucleic acid per mL before adding DNase I Buffer and rDNase I. In addition, rDNase I treatment can be divided into two steps. For details, see ''Perform routine DNase treatment'' on page 3 of the user manual.

If the sample cannot be diluted, simply increase the amount of rDNase I to 2-3 µL (4-6 U). Increasing the amount of enzyme may successfully remove contaminating DNA from samples containing up to 500 µg/mL nucleic acid in a 10-100 µL reaction. However, the efficacy of treating highly concentrated nucleic acid samples depends on the absolute level of DNA contamination, and residual DNA may or may not be detectable by PCR after 35-40 cycles.

Find additional tips, troubleshooting help, and resources within our Nucleic Acid Purification and Analysis Support Center.

We recommend that you include DNase treatment of all samples as a good practice, despite the fact that the majority of primers span across an intron and do not amplify (or in some cases, minimally amplify) contaminating genomic DNA. For more information see here.

Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Applications Support Center