Editing Capped mRNA (section)

=== Tail PCR ===

7. This section will append 120 Ts to the above synthesized Cre ORF with newly ligated 5′UTR and 3′UTR. Begin by diluting the PCR product (Cre ORF with 5′ UTR and 3′UTR) to 10 ng/μL.

<br/>8. Perform a 25 μL PCR reaction by combining the following:<br/>12.5 μL 2x KAPA HiFi Hotstart ReadyMix<br/>1 μL Tail Forward Primer (5 μM) – final concentration is 200 nM<br/>1 μL Tail Reverse T120 Primer (5 μM) – final concentration is 200 nM<br/>PCR product (10 ng/μL) – 10–20 ng<br/>H2O to bring final volume to 25 μl

<br/>9. Thermal cycler profile for Tail PCR reaction:<br/>95 °C 5 min<br/>98 °C 20 sec<br/>An external file that holds a picture, illustration, etc. Object name is nihms378773t3.jpg Object name is nihms378773t3.jpg<br/>25x<br/>65 °C 15 sec<br/>72 °C 45 sec<br/>72 °C 10 min<br/>4 °C Hold

<br/>10. Analyze the PCR product via gel electrophoresis (can use 1 μL of PCR reaction diluted 10x).

<br/>11. Perform QIAQuick Purification as described above in the Cre Recombinase ORF PCR amplification protocol.

<br/>12. Quantify and quality control the purification product by Nanodrop and dilute to 100 ng/μL