This resource is provided for those TURBO DNA-free customers that would prefer a procedure that allows a second DNA digestion step. Although a single DNase treatment is sufficient for the vast majority of DNA removal needs, Ambion recognizes that some users would appreciate the flexibility of a second treatment option. Sequential TURBO DNA-free treatments, however, require a special buffer for the second digestion step; the 10X TURBO DNase buffer provided in the kit cannot be used for this purpose. A unique digestion buffer for the second DNase reaction must be used to minimize the carryover of salt into the PCR step of RT-PCR. Otherwise, the limit of detection of targets in RT-PCR may be compromised.
Before you begin:
Prepare the 10X TURBO DNA-free Second Digest Buffer. This buffer contains: 200 mM Tris-HCl pH 7.5, 100 mM MgCl2, and 5 mM CaCl2, prepared with nuclease-free H2O. The final pH of the solution should be 7.5. If the pH is not 7.5, add NaOH or HCl as necessary, but do not introduce more than 50 mM of total monovalent salt to the 10X buffer when adjusting the pH in this way.
- After the DNase Inactivation Reagent treatment step in Section E.5 of the TURBO DNA-free manual, remove a volume of the RNA sample without disturbing the pellet, and transfer it to a new 0.5 ml tube.
- Add 0.1 volume of 10X TURBO DNA-free Second Digest Buffer, and 1 µl TURBO DNase to the RNA sample, mix gently, then continue with the standard protocol as described in Section E.2.
The RNA sample is now ready for downstream applications. However, Ambion recommends consideration of the following points:
a. For RT-PCR applications, we recommend that the volume of RNA that has been sequentially treated with TURBO DNA-free comprise no more than 20% of the final one-step RT-PCR reaction volume. If this amount of RNA passed into the RT-PCR reaction is too low, then consider increasing the RT-PCR volume to 50 µl or more, or performing a two-step reaction. For example, the RNA volume can be increased up to 40% of the final reverse transcription reaction volume in two-step applications.
b. Scientists at Ambion have observed that some RT-PCR amplicons are more sensitive to salt than others. Restricting the volume of the treated RNA sample to <20% can help to minimize these effects.