Editing In vitro transcription (section)

== Capped mRNA Synthesis ==

The recommended ratio of '''cap analog to GTP is 4:1.''' Cap analogs are sold separately.

1. Thaw the necessary kit components, mix and pulse-spin in a microfuge to collect solutions to bottom of tubes. Keep on ice.

2. Make '''20 mM''' GTP solution by combining 2 μl of 100 mM GTP and 8 μl of nuclease-free water.

Extra 20 mM GTP can be stored at –20°C for future use.

3. Prepare cap analog at the concentration of '''40 mM'''.&nbsp;



Assemble the reaction at room temperature in the following order:

Nuclease-free water X µl 10X

Reaction Buffer 2 µl

ATP (100 mM) 2 µl 10 mM final

UTP (100 mM) 2 µl 10 mM final

CTP (100 mM) 2 µl 10 mM final

GTP (20 mM) 2 µl'''2 '''mM final

Cap Analog (40 mM) 4 µl '''8 '''mM final

Template DNA X μl 1 µg

T7 RNA Polymerase Mix 2 µl

Total reaction volume 20 µl

5. Mix thoroughly, pulse-spin and incubate at 37°C for 2 hours





The yield per reaction is '''40–50 μg '''RNA with approximately '''80% capped''' RNA transcripts.

Table 1 shows the effect of varying the ratio of cap analog to GTP on the yield of RNA. Increasing the ratio of cap analog to GTP will increase the proportion of capped RNA transcripts, however it also significantly decreases the yield of the transcription reaction. A ratio of cap analog to '''GTP of 4:1 i'''s preferably used.

6. Optional: To remove template DNA, add 2 μl of DNase I (RNase-free), mix and incubate at 37°C for 15 minutes.

7. Proceed with purification of synthesized RNA or analysis of transcription products by gel electrophoresis.
[[Category:Protocols]]