Set up PCR on recent Sepia cDNA samples using both S. officianalis_rhodopsin_F, R primers & Sep_op_F2, R2 primers. PCR set up is here. Looking back at my old paper (gasp!) notebook from March 2007, I noticed that each primer set required differing amounts of Mg2+. Rhodopsin primers require 3mM Mg2+ in the PCR rxn and the opsin primers require 2mM Mg2+ in the PCR rxn. Mg2+ was added as required and is shown on the PCR set up link above.
Results:
Gel Loading (from left to right):
Opsin Primers (lanes 2-10), Rhodopsin Primers (same loading order, lanes 12-20)
1 - 100bp ladder
2 - retina
3 - fin
4 - 4th arm
5 - dorsal mantle center
6 - dorsal mantle side
7 - ventral mantle center
8 - ventral mantle side
9 - H2O
10 - H2O
Opsin Primers
We see an intense band in the retina sample, as expected, since opsin is constitutively expressed in this tissue. We also see a band in the fin and both dorsal mantle samples.
Negative controls are blank.
Rhodopsin Primers
We see an intense band in the retina sample. We also see a band in the fin sample. We see two bands in the ventral mantle center tissue, possibly suggesting a rhodopsin isoform is also being expressed in this tissue.
Negative controls are blank.
Overall, these results are rather intriguing because they clearly demonstrate that both genes are being differentially expressed in non-eye tissue of Sepia officianalis.