Additional RACE using gene specific primers (SR IDs: 1347 & 1348) for C.gigas COX2/PGS2 according to Clontech’s SMARTer cDNA RACE Kit protocol. 3’/5’ RACE cDNA libraries are from 20080619. Master mix calcs and set up is here. Cycling params followed “Program 2” of the Clontech protocol and are as follows:
25 cycles:
94°C 30 sec
68°C 30 sec
72°C 3 min
Reactions were run with both primers on both libraries, just to ensure that in case there was any confusion in primer design. When finished, I will remove 2uL of the PCR reaction for use in a nested PCR reaction. Will run a gel with both sets of products, once the nested PCR is completed.
Results:
Gel Layout:
Lane 1 - Hyperladder 1
Lanes 2-6 = 5’ RACE Library
Lane 2 - GSP1 (5’ RACE primer)
Lane 3 - GSP2 (3’ RACE primer)
Lane 4 - Neg. Control (no RACE primers)
Lane 5 - Neg. Control (GSP1, no Universal primer)
Lane 6 - Neg. Control (GSP2, no Universal primer)
Lane 7 - Empty
Lanes 8-12 = 3’ RACE Library
Lane 8 - GSP1 (5’ RACE primer)
Lane 9 - GSP2 (3’ RACE primer)
Lane 10 - Neg. Control (no RACE primers)
Lane 11 - Neg. Control (GSP1, no Universal primer)
Lane 12 - Neg. Control (GSP2, no Universal primer)
As has generally been the case, our primary RACE PCRs failed to produce any products. This is why I performed the nested PCR (described above) before viewing the results of this primary PCR.