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查看更多产品信息 TURBO DNase™ (2 U/μL) - FAQs (AM2239, AM2238)
16 个常见问题解答
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。
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.
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.
The TURBO DNAse itself is contained in a buffer containing glycerol, and therefore it will not freeze at -20°C. The reaction buffer is an aqueous salt solution which will remain stable even after multiple freeze/thaw cycles.
We do not recommend storing it under different conditions. You could also aliquot the DNAse, but under these circumstances it wouldn't be necessary.
Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Applications Support Center.
TURBO DNase does not contain DTT.
We do not offer the 10X Reaction Buffer that comes with TURBO DNase as a stand-alone item and the composition of the buffer is proprietary.
Yes, since the cleavage site for TURBO DNase is the phosphodiester bond, it cleaves both single- and double-stranded DNA.
The main difference between the two enzymes is that dsDNase is heat-labile (easily inactivated by moderate heat treatment (55 degrees C)), and hence does not need a separate heat inactivation step. It gets inactivated during the RT reaction (cDNA synthesis) itself. Turbo DNase on the other hand is not heat-labile and thus needs to be heat inactivated post-digestion or has to be removed by phenol-chloroform extraction.
Some bead carryover is common and has not been documented to affect downstream assays. The most common cause of bead carryover is too much input sample. The best way to determine if the sample amount is the cause of the issue is to run PBS with Xeno (artificial DNA/RNA) in the lysis buffer and then check if there is still bead carryover. If that is not the cause, the initial sample may require special processing, such as bead beating or liquification. If there is still bead carryover without the original sample, and in particular, if the carryover is present in the same wells for multiple runs, then this may be an instrument alignment issue.
Plastic consumables need to be purchased separately in order to use the MagMAX reagents on the MagMAX Express-96 or a KingFisher instrument. The particular consumables and the amount required will vary by kit and protocol so please check the user guide for the particular MagMAX kit to be sure that all required plastic consumables are available prior to sample processing.
The best place to download KingFisher Flex and KingFisher Duo Prime protocols for a particular MagMAX kit is the relevant MagMAX kit product page. The Bindlt script protocol (.bdz) files can be found under the Documents>Product literature section.
Yes, reaction volumes for MagMAX kits can be scaled up. In general, the amount of MagMAX reagents should be scaled up proportionally for the increase in sample input amount. Please see the specific kit user guide for any recommendation. If none are included, then optimization may be necessary.
Addition of BME (2-mercaptoethanol) to the lysis buffer of MagMAX kits is fine. This can be beneficial with more RNase-rich samples like certain types of tissues.
In general, we recommend adding 0.3 µL BME per 100 µL of lysate.
The TURBO DNase in the MagMAX mirVana Total RNA Isolation Kit is the same enzyme as the standalone TURBO DNase, but the concentrations of the enzyme differs and therefore it is not an exact equivalent. The TURBO DNase in the MagMAX mirVana Total RNA Isolation Kit is at 20 U/µL, whereas the standalone TURBO DNase is at 2 U/µL.
When using the MagMAX-96 for Microarrays Total RNA Isolation Kit modified spin protocol (https://www.thermofisher.com/us/en/home/references/protocols/nucleic-acid-purification-and-analysis/rna-protocol/isolating-small-rnaas-using-the-magmax-96-for-microarrays-kit.html) for isolating large + small RNA, we recommend going through the RNA isolation procedure and then treating the eluted RNA with TURBO DNase.