EnzymoPure™, This product is free from RNase, DNA exonuclease, and DNA endonuclease.
Stability And Storage
Store at -20℃.
Storage Temp
Store at -20°C
Shipped In
Ice chest + Ice pads
Grade
EnzymoPure™
Product Description
SP6 RNA Polymerase is a DNA-dependent RNA Polymerase that recognizes the SP6 promoter sequence high specifically and catalyzes the 5'→3' synthesis of RNA on either double-stranded or single-stranded DNA downstream from the SP6 promoter.SP6 RNA polymerase is useful for synthesizing large amounts of RNA suitable for in vitro translation and anti-sense RNA research. It also accepts modified nucleotides (e.g., biotin-, digoxigenin-, fluorescein-labeled nucleotides) as substrates to synthesize radiolabeled RNA probes.Please refer to Figure 1 for the effect of in vitro transcription catalyzed by Aladdin's SP6 RNA Polymerase using linearized plasmid DNA containing the SP6 Promoter as template. Figure 1. RNA transcripts synthesized by SP6 RNA Polymerase produced by aladdin and Competitor with linearized plasmid DNA containing SP6 Promoter as templates. Reactions were assembled as described in the protocol. Reaction system (20µl)
Application：
The SP6 RNA Polymerase is mainly used for RNA synthesis. The synthesized RNA can be used for hybridization, genomic DNA sequence analysis, RNase protection assay, antisense RNA synthesis, in vitro translation, RNA splicing studies, RNA secondary structure and RNA-protein interaction studies, nucleic acid amplification analysis, etc.
Source：
Recombinant SP6 RNA Polymerase expressed and purified from E. coli.Activity
Inactivation or inhibition：
SP6 RNA Polymerase can be inactivated by heating at 70℃ for 10 minutes or by adding appropriate amounts of EDTA. The activity of SP6 RNA Polymerase can be significantly inhibited by chelators and sodium, potassium or ammonium salts at concentrations higher than 150 mM.The SP6 consensus promoter sequence is ATTTAGGTGACACTATAGAAGNG, where G is the first base of transcription.
Precautions：
Keep SP6 RNA Polymerase on ice while handling, and store at -20℃ immediately after use.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.
Instructions for Use：
1. RNA Synthesisa. To prepare the linearized DNA template for transcription, digest the plasmid with appropriate endonucleases.b. Purify the DNA using phenol/chloroform extraction followed by ethanol precipitation, or using ’s DNA purification kit (#D0033).c. Set up the following reaction at room temperature:10X SP6 Transcription Buffer2µlNTP Mixture (10mM each)4µlLinearized DNA Template0.4-1µgRibonuclease Inhibitor (40U/µl)0.5µlSP6 RNA Polymerase (50U/µl)0.8-2µlDEPC-treated waterTo 20µld. Mix by gentle pipetting or vortex, spin briefly to collect all drops.e. Incubate at 37℃for 1~2h.f. Add 2µl of 0.5M EDTA (pH 8.0) or freeze at -20℃to terminate the reaction.g. Analyze the transcription product by electrophoresis or other appropriate methods.Notes: a) RNase contamination must be avoided. b) Due to the potential precipitation of DNA in the presence of spermidine, the reactions must be prepared at room temperature. c) Under the above reaction conditions, more than 10µg of RNA can be synthesized from 1µg of template DNA. d) Due to the high processivity of SP6 RNA polymerase, circular plasmid templates will generate long heterogeneous RNA transcripts in higher quantities compared to linear templates. Therefore, it is important to use completely linearized plasmid as template to ensure efficient synthesis of RNA transcripts of defined length. e) The above reaction can be scaled up or down as needed.2. Synthesis of radio-labeled RNAa. To prepare the linearized DNA template for transcription, digest the plasmid with appropriate endonucleases.b. Purify the DNA using phenol/chloroform extraction followed by ethanol precipitation, or using 's DNA purification kit (#D0033).c. Set up the following reaction at room temperature:10X SP6 Transcription Buffer2µl3 NTP Mixture (10mM each , without CTP)1µl100µM CTP2.4µl[α-32P]-CTP, ~30TBq/mmol(800Ci/mmol)1.85MBq(50µCi)Linearized DNA Template0.2~1µgRibonuclease Inhibitor (40U/µl)0.5µlSP6 RNA Polymerase (50U/µl)1µlDEPC-treated waterTo 20µld. Incubate at 37℃ for 1~2h.e. Freeze at -20℃ to terminate the reaction.f. Examine the labeling efficiency.Notes: a) Generally, the activity of RNA synthesized by the above method is 3-5 ×108 dpm/µg. b) NTPs labeled with [32P], [35S], or [3H] can also be used in the transcription reaction and the doses of reaction components should be adjusted accordingly. The recommended doses of labeled NTPs in a 20µl transcription reaction are as follows: 1.85MBq (50µCi) 5'-[α-32P]-CTP, ~30TBq/mmol(800Ci/mmol); 11.1MBq (300µCi) 5'-[α-35S]-UTP, ＞37TBq/mmol (＞1000Ci/mmol); 0.925MBq (25µCi) 5,6-[3H]-UTP, 1.1-2.2TBq/mmol (30-60Ci/mmol)。 c) The efficiency of full-length transcript synthesis decreases when the concentration of radioactively labeled NTP is below 12µM.3. Other applications such as RNA labeling with biotin and digoxigenin can be carried out by referring to the above protocol or relevant literature.
