High Yield RNA Synthesis Kit(＜2000nt)​

T7 RNA Polymerase is Isolated from E.coli carrying a plasmid which contains the T7 RNA Polymerase gene. T7 RNA Polymerase is a DNA-dependent RNA polymerase that is highly specific for the T7 phage promoters.

Components of the product 

Storage Condition

All components should be stored at -20°C

Unit Definition

One unit is defined as the amount of enzyme that will incorporate 1 nmol ATP into acid-insoluble material in a total reaction volume of 50 µl in 1 hour at 37°C in 1X RNA Polymerase Reaction Buffer.。

Quality Control

Exonuclease Activity: Incubation of a 40 µl reaction containing a minimum of 40 units of T7 RNA Polymerase with 2.5 μg λ-Hind III digest DNA for 16 h at 37℃ results in no detectable degradation as determined.

Endonuclease Activity: Incubation of a 50 µl reaction containing a minimum of 50 units of T7 RNA Polymerase with 1 μg λDNA for 16 h at 37℃ results in no detectable degradation as

determined.

RNase Activity: Incubation of a 40 µl reaction containing a minimum of 40 units of T7 RNA Polymerase with 1 μg MS2 RNA for 2 h at 37 ℃ results in no detectable degradation as determined.

DNase Activity: Incubation of a 40 µl reaction containing a minimum of 40 units of T7 RNA Polymerase with 4 μg pUC19 DNA for 16 h at 37℃ results in no detectable degradation as determined.

Protocol

1. Assemble the reaction at room temperature in the following order:

Standard RNA Synthesis：

RNA Synthesis with Modified Nucleotides：

2. Mix thoroughly, spin briefly to collect all drops and incubate at 37°C for 2 h.

3. To stop the reaction, add 2 µL 0.2M EDTA (pH=8.0@25℃) to the reaction system or heat the reaction system at 75℃ for 20 min.

3.Add 1μL of DNase I to the reaction system and incubate at 37℃ for 15min to digest the DNA template.

Precaution

• For your safety and health, please wear lab coat and disposable gloves.

• The reaction system should be prepared at room temperature to avoid precipitation of DNA and spermidine at 4°C.

• If the transcription of the template produces full-length RNA, but the yield is significantly lower than expected, it may be that the contaminants in the DNA template inhibit the activity of the RNA polymerase, or the DNA concentration may not be correct.

• The volume of the reaction system can be scaled up or down according to the actual demand.