DuraScribe® SP6 Transcription Kit
DuraScribe® SP6 Transcription Kit
The DuraScribe® T7 and SP6 Transcription Kits* produce 2´-fluorine-modified RNA transcripts—called DuraScript® RNA—that are completely resistant to RNase A (Fig. 1).1 DuraScript RNA is more stable in storage and in all RNA applications, and can be made and used without the tedious procedures normally required for keeping RNA transcripts intact. Research indicates that double-stranded DuraScript RNA can be delivered into cultured mammalian cells in the presence of serum and without the need for transfection reagents.2 Double-stranded oligonucleotides, linearized plasmids, or PCR products with a T7 or SP6 promoter can be transcribed in a DuraScribe transcription reaction.
The DuraScribe T7 and SP6 RNA Polymerases provided in the kits efficiently incorporate 2´-Fluorine-CTP (2´-F-dCTP) and 2´-Fluorine-UTP (2´-F-dUTP), as well as ATP and GTP into full length transcripts. The standard 20-µl DuraScribe reaction, using 1 µg of DNA template, produces approximately 50 µg of DuraScript RNA (Fig. 2). The DuraScribe T7 and SP6 RNA Polymerases recognize the same T7 and SP6 transcription promoters as standard T7 and SP6 RNA Polymerases.
Figure 1. DuraScript® RNA is resistant to RNase A digestion. A 1.4-kb standard RNA transcript and a 1.4-kb DuraScript RNA transcript were each incubated with 1 U of highly purified RNase A for 30 minutes. The standard RNA transcript was completely degraded while the DuraScript RNA transcript remained intact. Lane M, size ladder; lane 1, 1.4-kb standard RNA transcript; lane 2, standard RNA after RNase A treatment; lane 3, 1.4-kb DuraScript RNA; lane 4, DuraScript RNA after RNase A treatment.
Figure 2. Yield of RNA from a DuraScribe® T7 Transcription reaction. A standard reaction (4-6 hours) produced 40-60 µg of a 1.4-kb DuraScript® RNA.
Figure 3 (click to enlarge). DuraScribe® T7 and SP6 RNA Polymerases efficiently incorporate 2´-F-dCTP and 2´-F-dUTP into full length DuraScript® RNA. The presence of the fluorine at the 2´-position of the 2´-F-dC and 2´-F-dU nucleotides prevents RNase A digestion. The result is DuraScript® RNA that is completely resistant to RNase A and related ribonucleases.
|Figure 4. DuraScript® RNA is completely resistant to "finger" nucleases. A 1.4-kb standard RNA transcript and a 1.4-kb DuraScript RNA transcript were produced using sterile water or water that had been contaminated by exposure to the hands of a test subject. The standard RNA shows extensive degradation from "finger" nucleases in the contaminated water while the DuraScript RNA remains fully intact. M, Size ladder; Lane 1, Standard RNA transcript; Lane 2, Standard RNA after "finger" nuclease exposure; Lane 3, DuraScript RNA; Lane 4, DuraScript RNA after "finger" nuclease exposure.||Figure 5. DuraScript® RNA is stable in tissue culture media for at least 2 hours. Five micrograms of a 1.4-kb standard RNA transcript and a DuraScript RNA transcript were incubated in 100 µl of tissue culture media (D-MEM + 10% fetal calf serum) at 37°C. Lane 1, RNA ladder; Lane 2, standard RNA; Lane 3, standard RNA after 15 minutes in tissue culture media; Lane 4, DuraScript RNA; Lanes 5-8 DuraScript RNA after 15, 30, 60, and 120 minutes, respectively, in tissue culture media.|
- Capodici, J. et al. (2002) J. Immunol. 169, 5196.
- Meis, J. and Chen, F. (2002) EPICENTRE Forum 9(1), 10.
* Covered by issued and/or pending patents.
|DuraScribe® T7 Transcription Kit|
|Contents: DuraScribe® T7 Enzyme Mix, RNase-Free DuraScribe® T7 10X Reaction Buffer, ATP, GTP, 2´-F-dCTP, 2´-F-dUTP, DNase I, DTT, Control Template DNA (linearized), Sterile Deionized Water.|
|DuraScribe® SP6 Transcription Kit|
|Contents: DuraScribe® SP6 Enzyme Mix, DuraScribe® SP6 10X Reaction Buffer, ATP, GTP, 2´-F-dCTP, 2´-F-dUTP, DNase I, DTT, Control Template DNA (linearized), Sterile Deionized Water.|