Preparation of template dna

Figure 2 PCR prime example
T7 +1
5’-TAATACGACTCACTATAGGGtgaattgcgcaatactgactgg-3’
The +1 base (in bold) is the first base incorporated in RNA during transcription. The underline shows the minimum promoter sequence needed for efficient transcription. The lower-case sequence is gene-specific sequence.
 
mRNA Synthesis Protocols
We strongly recommend wearing gloves and using nuclease-free tubes and reagents to avoid RNase contamination. Reactions are typically 20 μL but can be scaled up and down as needed. Reactions should be assembled in nuclease-free microfuge tubes or PCR strip tubes.
1. Assemble transcription reaction at room temp.
The spermidine in the 5× Reaction Buffer can coprecipitate the template DNA if the reaction is assembled on ice. Add the 5× Reaction Buffer after the water and the ribonucleotides are already in the tube.
The following amounts are for a single 20 μL reaction. Reactions may be scaled up or down if desired.

Component	Amount
Nuclease-free Water	to 20 μL
2× ARCA/NTP Mix	10 μL
Template DNA	1 μg*
5× Reaction Buffer	4 μL
Enzyme Mix	1 μL

*The transcript may vary depending on the amount of template DNA.
 
2. Mix thoroughly
Gently flick the tube or pipette the mixture up and down gently, and then microfuge tube briefly to collect the reaction mixture at the bottom of the tube.
 3. Incubate at 37°C, 2 hr.
Typically, 2 hours of incubation is sufficient. Incubation time can be extended to 4h for maximum yield.
 4. (optional) Add 1 μL DNase (RNase-free), mix well and incubate 30 min at 37°C.
This DNase treatment removes the template DNA. For many applications it may not be necessary because the template DNA will be present at a very low concentration relative to the RNA.
a. Add 1 μL DNaseI,and mix well (the reaction may be viscous).
b. Incubate at 37°C for 30 min.
 5. Poly(A) tailing procedure. Set up the tailing reaction as below. The IVT reaction solution does not need to be purified.