---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
layout: post
title: PCR - Crassostrea gigas and sikamea Mantle gDNA from Marinelli Shellfish Company
date: '2019-12-03 15:30'
tags:
  - PCR
  - gel
  - Crassostrea gigas
  - Crassostrea sikamea
  - Pacific oyster
  - Kumamoto oyster
  - gDNA
categories:
  - 2019
  - Miscellaneous
---
I ran this PCR a couple of times before and, embarrassingly, [I had ordered/used the wrong primers](https://robertslab.github.io/sams-notebook/posts/2019/2019-11-21-PCR---Crassostrea-gigas-and-sikamea-Mantle-gDNA-from-Marinellie-Shellfish-Company---No-Multiplex/).

Well, I ordered the correct universal cytochrome oxidase primers and used those!

| SR ID | Primer Name | Sequence                   |
|-------|-------------|----------------------------|
| 1739  | HC02198     | taaacttcagggtgaccaaaaaatca |
| 1738  | LCO1490     | ggtcaacaaatcataaagatattgg  |
| 1736  | COCsi546r   | AAGTAACCTTAATAGATCAGGGAACC |
| 1735  | COCgi269r   | TCGAGGAAATTGCATGTCTGCTACAA |


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).](https://www.researchgate.net/profile/Ximing_Guo/publication/259643859_Identification_of_Crassostrea_ariakensis_and_related_oysters_by_multiplex_species-specific_PCR/links/55c79eb708aeb9756746e35e/Identification-of-Crassostrea-ariakensis-and-related-oysters-by-multiplex-species-specific-PCR.pdf)

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.](https://www.researchgate.net/publication/15316743_DNA_primers_for_amplification_of_mitochondrial_Cytochrome_C_oxidase_subunit_I_from_diverse_metazoan_invertebrates)


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)        |
|------------------------|----------------------|-----------|---------------------------|
| DNA                    | 4                    | NA        | NA                        |
| 2x Apex Master Mix | 12.5                 | 18        | 225                       |
| HC02198 (100uM)              | 0.15                 | 18        | 2.7                       |
| LCO1490 (100uM)           | 0.15                 | 18        | 2.7                       |
| COCgi269r (100uM)           | 0.1                  | 18        | 1.8                       |
| COCsi546r (100uM)            | 0.1                  | 18        | 1.8                       |
| H2O                    | 8                    | 18        | 144                       |
|                        | 25                   |           | Add 21uL to each PCR tube |


Cycling params:

95<sup>o</sup>C for 10mins

30 cycles of:

- 95<sup>o</sup>C 1min
- 51<sup>o</sup>C 1min
- 72<sup>o</sup>C 1min

72<sup>o</sup>C 10mins

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:

![GeneRuler DNA Ladder Mix](https://github.com/RobertsLab/resources/blob/master/protocols/Commercial_Protocols/ThermoFisher_OgeneRuler_DNA_Ladder_Mix_F100439.jpg?raw=true)


---

# RESULTS


![Gel image](https://github.com/RobertsLab/sams-notebook/blob/master/images/20191203_gel_cgigas_csikamea_PCR.jpg?raw=true)



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`C.gigas 1911SS` samples


Now, this is where things get interesting...

In the `C.sikamea CA5SS`:

- 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).

In the `C.sikamea 1911SS`:

- 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)


![Confusing bag label marking oysters as Kumamoto and Pacific oyster](https://github.com/RobertsLab/sams-notebook/blob/master/images/20191030_marinelli_shellfish-04.jpg?raw=true)
