PCR - Crassostrea gigas and sikamea Mantle gDNA from Marinellie Shellfish Company - No Multiplex

UPDATE 20191125

Since the results I obtained on my final attempt to get this to work failed, I decided to double-check the primer sequences.

Well, I ordered/used the wrong sequences! The two general Crassostrea spp. primers ordered were the 28s primers listed in that paper, instead of the cytochrome oxidase primers! I’ve ordered the correct universal CO primers [which are actually listed in this paper: (Folmer, O., M. Black, W. Hoeh, R. Lutz & R. Vrijenhoek. 1994. DNA primers for amplification of mitochondrial cytochrome C oxidase subunit I from diverse metazoan invertebrate. Mol. Mar. Biol. Biotechnol. 3:294–299.) and will re-run this.

I’m leaving the original post below for posterity.

Primers and cycling parameters were taken from this publication:

SR ID Primer Name Sequence

Instead of running a multiplex PCR as before, I ran each set of species-specific primer pairs independently.

The COforward/reverse primers should amplify any Crassostrea spp. DNA (i.e. a positive control - 697bp) and the other two primers will amplify either C.gigas (Cgi269r - 269bp) or C.sikamea (Csi546r - 546bp).

Master mix calcs:

Component Single Rxn Vol. (uL) Num. Rxns Total Volumes (uL)
2x Apex Master Mix 12.5 18 225
COforward (100uM) 0.15 18 2.7
reverse primer (100uM) 0.10 18 1.8
H2O 8.25 18 148.5
  25   Add 21uL to each PCR tube

Cycling params:

95oC for 10mins

30 cycles of:

  • 95oC 1min
  • 51oC 1min
  • 72oC 1min

72oC 10mins

Used the GeneRuler DNA Ladder Mix (ThermoFisher) for all gels:

GeneRuler DNA Ladder Mix


GEL: C.gigas-specific primers:

Gel image from C.gigas-specific primers

GEL: C.sikamea-specific primers:

Gel image from C.sikamea-specific primers

GEL: Crassostrea spp. primers:

Gel image from Crassostrea species primers

Well, it looks like the two species-specific reverse primers don’t seem to work with the Crassostrea spp. forward primer.

However, the Crassostrea spp., despite the fact that they function properly (in that they amplify in all Crassostrea DNA), they don’t produce the expected band size that’s mentioned in the paper (697bp). Talking with Steven, I don’t think we’re going to pursue much more of this.