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.