FAQ: How can I improve the yield of RNA when using the HiScribe™ T7 mRNA Kit with CleanCap® Reagent AG?

Make sure that the NTPs, CleanCap Reagent AG, 10X Buffer and double-stranded DNA template are thawed and kept at room temperature during reaction set up (keep the T7 RNA Polymerase Mix on ice). Make sure that these components (NTPs, CleanCap® Reagent AG, 10X Buffer and dsDNA template) are mixed well prior to use by gently vortexing for 2-3 seconds and spinning down in a microfuge to collect the liquid at the bottom of the tube  Set the reaction up at room temperature in the order listed in the manual.  Gently mix the reaction by pipetting up and down and briefly spin in a microfuge before moving the reaction at 37°C.

In addition, make sure to use RNase-free tubes and filter tips, and always wear gloves when handling the reagents and reactions.  Make sure that your DNA template is very clean and verify that the concentration is correct.