Ran PCR to amplify full-length cDNAs of PGS1 & PGS2 (COX1 & COX2) using primers designed to anneal in the 5’/3’UTRs of each isoform. PGS1 primers = SRIDs: 1377, 1378. PGS2 primers = 1376, 1375. Master mix calcs and cycling params are here. cDNA was pooled cDNA made 20110311 from various tissues.
PGS1 Expected Size = ~2300bp
PGS2 Expected Size = ~2500bp
Lane 1 – Hyperladder I (Bioline)
Lane 2 – PGS1
Lane 3 – PGS1 NTC
Lane 4 – PGS1 NTC
Lane 5 – PGS2
Lane 6 – PGS2 NTC
Lane 7 – PGS2 NTC
PGS1 Results: PGS1 PCR produces a single band of the expected size (~2300bp), indicating that the two primers, which were designed to anneal in the 5’/3’UTRs of the gene and should be highly specific to just this isoform, work perfectly. The band was excised and stored @ -20C in “Sam’s Miscellaneous” box.
PGS2 Results: PGS2 PCR didn’t produce any product. Will repeat with a lower annealing temp (50C instead of 55C).