TURBO DNA-free™ 试剂盒

可以，虽然单次DNase处理通常可以满足绝大部分DNA去除需求，但也可以选择进行二次处理。请点击此处（https://www.thermofisher.com/us/en/home/references/ambion-tech-support/nuclease-enzymes/turbo-dna-free-second-digest-protocol.html），查看实验方案。

以下方法可去除DNase：酸性苯酚：氯仿抽提、氯化锂沉淀、EDTA和热灭活，或使用我们的MegaClear 转录回收试剂盒（货号AM1908）。
此外，我们的Ambion TURBO DNA-free试剂盒（货号AM1907）包含DNase灭活试剂，可用于去除DNase以及镁和钙等二价阳离子，这些物质在RNA随样品一起加热时可催化RNA降解。

Yes, while a single DNase treatment is typically sufficient for the vast majority of DNA removal needs, a second treatment option is available. Please note that sequential TURBO DNA-free treatments 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.

Please see the second digest protocol below:

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.

Protocol:
After the DNase Inactivation Reagent treatment step in Section E.5 of the TURBO DNA-free Kit manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/1907M_turbodnafree_UG.pdf), 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, we recommend consideration of the following points:

- 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 the 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.

- We have observed in-house 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.

DNase can be removed by the following methods: acid phenol:choloform extraction, lithium chloride precipitation, EDTA, and heat inactivation, or using our MegaClear Transcription Clean-Up Kit (Cat. No. AM1908).

Alternatively, our Invitrogen TURBO DNA-free Kit (Cat. No. AM1907) is supplied with a DNase inactivation reagent that can be used to remove the DNase, as well as divalent cations such as magnesium and calcium, which can catalyze RNA degradation when RNA is heated with the sample.

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.