- Hyperactive TURBO DNase is a catalytically superior enzyme compared to wild type DNase I
- Removes trace quantities of DNA that can plague RT-PCR
- Reagent included to completely remove DNase without phenol treatment or heating
TURBO DNA-free: The Best Method for Genomic DNA Removal prior to RT-PCR
When Ambion introduced the highly potent TURBO DNase™ (patent pending) in the TURBO DNA-free Kit, a new standard was set in DNA removal capabilities. TURBO DNase is a recombinant, engineered form of DNase I that is much more efficient than wild type DNase I in digesting away trace amounts of unwanted DNA. TURBO DNase binds DNA substrates 6-fold more tightly than traditional DNase I, making this enzyme the tool of choice for clearing residual DNA that can generate a false positive signal in RT-PCR applications.
Removal of DNA Further Enhanced by >600-fold
Ambion's continuing commitment to developing the best possible DNA removal technology has produced an enhancer that now increases the effectivness of TURBO DNase by two orders of magnitude (upper figure at right). This enhancer is now available as part of the improved version of the TURBO DNA-free Kit.
Since the TURBO DNase in the TURBO DNA-free Kit is already extremely potent, we had to create an RNA preparation that was largely comprised of genomic DNA to quantify the level of improvement offered by the new DNase enhancer (see bottom figure at right). Consequently, we prepared a mouse spleen total RNA sample that contained 70% DNA and only 30% RNA. This DNA-laden RNA was then treated with either the orignal TURBO DNA-free Kit or with the improved version. To better evaluate the differences in DNA removal between the two kits, we modified the standard protocol by using four times less TURBO DNase than is normally used. This was necessary to produce a detectable, residual DNA signal in realtime PCR by both kit formulations, so that their differences in fold DNA removal could be accurately calculated. Using these conditions, the original formulation of TURBO DNA-free reduced the genomic DNA (gDNA) that could be PCR amplified by 451-fold. The improved kit reduced the gDNA contamination much further, by 277,000-fold, or 614 times more than the kit without the enhancer.
Efficient DNase and Divalent Cation Removal Without Organic Extraction or Precipitation
Conventional DNase treatment procedures of RNA samples prior to RT-PCR typically call for inactivation of the DNase by phenol:CHCl3 extraction or heating followed by a precipitation step to concentrate the RNA. Phenol:CHCl3 extractions can be cumbersome and time-consuming, especially when large numbers of samples are involved. Heating the sample to inactivate DNase can lead to chemical degradation of the RNA by divalent cations present in the DNase buffer (Figure 2 at right, lane 5). The TURBO DNA-free Kit circumvents these problems using a novel DNase Inactivation Reagent. In addition to removing the TURBO DNase from the reaction, the Inactivation Reagent also binds and removes divalent cations from the TURBO DNase Reaction Buffer or carried over from the RNA sample (Figure 2).
A Complete Kit for DNase Treatment and Removal
The TURBO DNA-free Kit comes with all the reagents needed to perform 50 TURBO DNase I treatments. The DNase Inactivation Reagent provided with the kit is based on a new formulation that allows for easier handling and removal prior to RT-PCR. As with the DNA-free Kit, the Inactivation Reagent effectively removes DNase and divalent cations from the reaction in a single step without phenol extraction or heating. A specially formulated TURBO DNase 10X Reaction Buffer is also provided.
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