Editing Capped mRNA (section)

=== UTR Ligation ===

5. Ligate the UTRs to the CRE recombinase ORF amplicon isolated above. Begin by combining the following for a 20 μL reaction:<br/>4 μL 5′ UTR (1 μM)<br/>4 μL 3′ UTR-Phosphorylated (1 μM)<br/>2 μL 5′ Splint (1 μM)<br/>2 μL 3′ Splint (1 μM)<br/>2 μL 10x Buffer<br/>1 μL Ampligase<br/>1 μL CRE ORF amplicon<br/>4 μl Water<br/><br/>6. Thermal profile for UTR Ligation:<br/>95 °C 10 sec<br/>An external file that holds a picture, illustration, etc. Object name is nihms378773t2.jpg Object name is nihms378773t2.jpg<br/>5 cycles<br/>45 °C 60 sec<br/>50 °C 60 sec<br/>55 °C 60 sec<br/>60 °C 60 sec<br/>4 °C Hold

Perform QIAQuick Purification as described above in the Cre Recombinase ORF PCR amplification protocol.

NOTE: It may be desirable to TA clone the newly generated UTR-flanked Cre amplicon into a plasmid to avoid the need to re-amplify from the Cre DNA plasmid. This can be accomplished by using the pcDNA3.3-TOPO TA Cloning Kit.

NOTE: Check and ensure the ligation worked by diagnostic PCR by using flanking primers and subsequently performing gel electrophoresis to double-check if the product is of the expected size. This is a critical juncture for troubleshooting—if the ligation is unsuccessful (it often requires multiple attempts) try either a wider/smaller gradient of temperatures or try a sequential ligation. The difficulty lies in that the two splints usually have different annealing conditions so both will not stick at once.