I ran this PCR a couple of times before and, embarrassingly, I had ordered/used the wrong primers.
Well, I ordered the correct universal cytochrome oxidase primers and used those!
|SR ID||Primer Name||Sequence|
Primers and cycling parameters were taken from this publication:
- Haiyan Wang and Ximing Guo “Identification of Crassostrea ariakensis and Related Oysters by Multiplex Species-Specific PCR,” Journal of Shellfish Research 27(3), 481-487, (1 May 2008).
Universal cytochrome oxidase primers were from 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.
This is a multiplex PCR, where the HC02198 and LCO1490 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|
|25||Add 21uL to each PCR tube|
95oC for 10mins
30 cycles of:
- 95oC 1min
- 51oC 1min
- 72oC 1min
PCR reactions were run on a 1.5% agarose, 1x low TAE gel with ethidium bromide.
Used the GeneRuler DNA Ladder Mix (ThermoFisher) for all gels:
Alrighty, this is what we expected to see (at least in terms of primer functionality). We see:
A band of ~700bp in all samples (i.e. the universal cytochrome oxidase primers)
A band of ~270bp in the
Now, this is where things get interesting…
A prominent band of ~550bp, indicating these are C.sikamea
A less prominent, but definitive, band at ~270bp, indicating the presence of C.gigas cytochrome oxidase sequence. Suggests that this particular stock has hybridized with C.gigas (or, there was some sort of cross contamination of DNA/tissue; unlikely as we don’t see potential cross contamination of C.sikamea DNA in the other samples).
- A prominent band of ~270bp, indicating these are C.gigas, despite the bag label indicating them as Kumamoto and/or Pacific oysters (see image below)