In addition to the above techniques, researchers now have a new and convenient option for removal of DNA and DNase I from RNA samples. Ambion's DNA-free™ DNase Treatment and Removal Reagents are designed for the removal of contaminating DNA from RNA samples and for the removal of DNase after treatment. As described above, DNase is typically inactivated by heat treatment, and can also be removed from treated preps by phenol extraction. Heat inactivation can present problems, however, as the temperature at which DNase is inactivated also catalyzes RNase-independent RNA strand scission in the presence of divalent cations. Phenol extraction is also avoided by researchers who do not want to work with phenol, or who worry about sample loss.
DNA-free avoids both methods of DNase I inactivation by supplying a novel DNase Removal reagent that effectively removes DNase and divalent cations from the reaction mixture. The DNase/cation removal step takes a mere three-minute incubation. No organic extraction, EDTA addition or heat inactivation is required.
The DNA-free DNase Treatment and Removal Reagents are provided with RNase-free DNase I, an optimized 10X Reaction Buffer, and the DNase Removal Reagent. The DNA-free Reagents are now also part of the RNAqueous®-4PCR Kit, combining the features and benefits of RNAqueous with those of DNA-free.
| Figure 4. RT-PCR Experiments Using Total RNA DNase-Treated Using DNA-free Reagents. Five µl of RNA samples isolated using Ambion's RNAqueous® Kit were used as templates for reverse transcription reactions; 10% of the resulting cDNA was amplified by PCR using S15 primers. The lanes to the left of the markers are PCR reactions done without reverse transcription, demostrating the lack of genomic DNA contamination in these RNA samples. The lanes to the right of the markers show the S15 RT-PCR product from the indicated samples. |
In addition to DNA-free, Ambion offers many quality products to facilitate successful RT-PCR experiments. These include RNase-free pipette tips and PCR tubes, RNase-free DNase I, ToTALLY RNA, RNAqueous, and Poly(A)Purist RNA Isolation Kits, RETROscript First Strand cDNA Synthesis Kit, and SuperTaq™ thermostable DNA polymerase. All of Ambion's products designed for use with RNA undergo rigorous quality testing and are certified RNase-free.
The Polymerase Chain Reaction (PCR) is covered by patents owned by Hoffman-LaRoche. Use of the PCR process requires a license. A license for research may be obtained by purchase and use of authorized reagents and DNA thermal cyclers.
SuperTaq™ is made by Enzyme Technologies Limited and sold under licensing arrangements with F. Hoffmann-La Roche Ltd., Roche Molecular Systems, Inc. and the Perkin-Elmer Corporation. Ambion is a distributor of Enzyme Technologies Limited.
Purchase of SuperTaq is accompanied by a limited license for its use in the Polymerase Chain Reaction (PCR) and RT-PCR process for research in conjunction with a thermal cycler, the use of which in the automated performance of the PCR and RT-PCR process is covered by the up-front license fee, either by payment to Perkin-Elmer, or as purchased, i.e., an authorized thermal cyler.
Super Taq is not available for sale directly from Ambion in the United Kingdom, France, BeNeLux, Denmark, Sweden, Italy, Austria, Switzerland, Singapore, and Taiwan. Contact Enzyme Technologies LTD, Unit 4, 61 Ditton Walk, Cambridge CB5 8QD, U.K. (phone 44-1223-412-583) for more information.
