DuraScribe® SP6 Transcription Kit

DuraScribe™ Transcription Kit

* The use of DuraScribe™ T7 Transcription Kit to synthesise nucleic acids with non-canonical bases or for partial ribosubstitution is covered by U.S. patents 5,849,546; 6,107,037 and other patents issued or pending.
These products are accompanied by a limited non-exclusive license for the purchaser to use the purchased product(s) solely for life science research. Contact Epicentre® concerning licenses for other uses.

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Last modified: 2008-07-24 11:28:14

The DuraScribe® T7 and SP6 Transcription Kits produce 2'-Fluorine-modified RNA transcripts called DuraScript® RNA that are completely resistant to RNase A. DuraScript® RNA is more stable in storage and in all RNA applications and can be made and used without the worry and time-consuming procedures normally required for keeping RNA transcripts intact. Recent 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. 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, are mutant forms of T7 and SP6 RNA Polymerases that 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.(Figure 1) The standard 20-µl DuraScribe® reaction, using 1µg of DNA template, produces approximately 50µg of DuraScript® RNA. The DuraScribe® T7 RNA Polymerase recognizes the same T7 transcription promoters as standard T7 RNA Polymerase.

Figure 1. 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. The presence of the 2'-F-dC and 2'-F-dU nucleotides render DuraScript RNA completely resistant to the ubiquitous RNase A. DuraScript RNA retains sensitivity to RNase T1 and RNase H. DuraScript RNA is resistant to DNase I degradation so that DuraScribe reactions can be treated to remove the DNA template after the transcription reaction is complete. Double-stranded DuraScript RNA can be digested by RNase III.

	Figure 2. 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. 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 3. 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 4. 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.

Last modified: 2008-05-14 16:46:46

Applications

• In situ hybridisation
• Ribonuclease protection assays
• RNA interference (RNAi)
• Competitive RT-PCR assays
• Ribozyme and aptamer studies
• Anti-sense RNA

Benefits

• DuraScript® RNA is completely resistant to RNase A, nucleases found on the human skin
• A standard DuraScribe reaction produces 50µg of DuraScript® RNA.
• DuraScript® RNA has improved stability in storage and most applications
• No need for gloves, DEPC-treatment of reagents, or RNase Inhibitors when making or working with DuraScript® RNA.
• DuraScribe® T7 and SP6 RNA Polymerases utilize the same transcription promoters as standard T7 and SP6 RNA Polymerases.
• Double-stranded DuraScript® RNA is taken up by cultured mammalian cells in the presence of serum and without the need for transfection reagents.
• DuraScript® RNA can be digested by RNase T1 and RNase H, and double-stranded DuraScript® RNA can be digested by RNase III.
• Fluorescently-labeled DuraScribe® RNA can be readily produced.

Figure 5. Forty to sixty µg of a 1.4-kb DuraScript® RNA is produced in a standard 4-hour DuraScribe® T7 Transcription reaction.	Figure 6. A DuraScribe® reaction can be scaled-up to produce even more DuraScript® RNA. Shown is scale up of a DuraScribe T7 Transcription reaction.

* The use of DuraScribe® Transcription Kits to synthesize nucleic acids with non-canonical bases or for partial ribosubstitution is covered by U.S. patents 5,849,546; 6,107,037 and other patents issued or pending. These products are accompanied by a limited on-exclusive license for the purchaser to use the purchased product(s) solely for life science research. Contact EPICENTRE® concerning licenses for other uses.

Note: EPICENTRE® products are licensed under U.S. and international patent rights owned by the Carnegie Institution of Washington that cover RNA interference. These products are accompanied by a limited non-exclusive worldwide license under the Carnegie Institution of Washington's patent rights for researchers at academic or other not-for-profit institutions to use the product for non-profit research. However, use of dsRNA for RNA interference by for-profit organizations requires a license from the Carnegie Institution of Washington. For-profit institutions should contact Gloria Brienza of the Carnegie Institution of Washington, 1530 P Street, N.W., Washington, D.C. 20005-1910. E-mail: gbrienza@pst.ciw.edu.

Last modified: 2008-05-14 16:46:46

Protocols for: DuraScribe® T7 & SP6 Transcription Kits

(catalogue number DS010910 / DS10925 / DS041010)

Please note: all protocols off site are the responsibility of the products supplier

Last modified: 2008-05-14 16:46:46

References

1. Meis, J.E. and Chen, F. (2002) EPICENTRE Forum 9 (1), 10.
2. Capodici, J. et al. (2002) J. Immunol. 169, 5196.

Last modified: 2008-05-14 16:46:46