Hello,
I have performed nucleic acid extraction using the CTAB protocol, and in the final step, I eluted the material with 50 ul of RNase-free ddH2O. A portion of this material was subjected to DNAse treatment. Currently, I have 30 ul of the extracted material remaining and would like to inquire about the possibility of using RNAse A from the "NucleoSpin Plant II, Mini kit for DNA from plants" by MACHEREY‑NAGEL to treat DNA that has already been extracted with the CTAB protocol.
The RNAse A from this kit is provided as 30 mg of lyophilized material, which should be resuspended in 2.5 ml of ddH2O, resulting in a concentration of 12 mg/ml.
I would greatly appreciate guidance on whether it is suitable to use this specific RNAse A for treating DNA already extracted using the CTAB protocol. If it is feasible, I kindly request information on the quantity to be added for effective treatment.
Thank you for your expertise and assistance.
Best regards,
Hello Miriam Negussu
Yes, you may use RNAse A for treating DNA which has already been extracted. You may add 50μg/ml of RNase A and incubate the mixture for 1 hour at 37 degree C. The treatment of DNA with RNase A should be done in Tris buffer at the end of the extraction protocol. Salting out step can be repeated as before according to the protocol which you have followed to obtain DNA.
The DNA pellet may be dissolved in Tris-EDTA for DNA protection from degradation by metal dependent nucleases during storage.
Best.
Hello Malcolm Nobre ,
I wanted to express my gratitude for your prompt response. In the kit's manual from which the RNAse A originated, it recommends using 10 µL directly on the powder and the extraction buffer (400 µL). Consequently, I calculated that the manufacturer's recommended final concentration is approximately 300 µg/mL.
Therefore, would it suffice to use 50 µg/µL on the extracted DNA? I hope my question is clear.
Hi Miriam,
The RNAse leaflet (https://www.mn-net.com/media/pdf/0a/9b/e1/LF-RNase-A.pdf) does not specify the capability of the enzyme (typically given as U/unit - the amount required to produce 1 nmol of ribonucleotide). However, you can obviously try to use it outside of the kit, but the amount required is practically your best guess. Use Qubit/Bioanalyzer if you can to measure RNA cc before/after treatment.