An additional attempt to get the actin primers to work for use in screening samples for bay/sea scallop hybrids. The scallop_actin_fw primer was used in conjunction with the following:

bay_actin_Rv0 (Rxn 1)

bay_actin_Rv2 (Rxn 2)

sea_actin_Rv4 (Rxn 3)

sea_actin_Rv5 (Rxn 4)

PCR set up is here. Just used Bay or Sea scallop gDNA (chelexed). When/If get this working correctly, will start screening the hybrid samples. Anneal of 53C.

Lane 1 – 100bp Ladder

Lane 2 – Rxn 1: Bay

Lane 3 – Rxn 1: Sea

Lane 4 – Rxn 1: H2O

Lane 5 – Rxn 1: H2O

Lane 6 – Rxn 2: Bay

Lane 7 – Rxn 2: Sea

Lane 8 – Rxn 2: H2O

Lane 9 – Rxn 2: H2O

Lane 10 – Rxn 3: Bay

Lane 11 – Rxn 3: Sea

Lane 12 – Rxn 3: H2O

Lane 13 – Rxn 3: H2O

Lane 14 – Rxn 4: Bay

Lane 15 – Rxn 4: Sea

Lane 16 – Rxn 4: H2O

Lane 17 – Rxn 4: H2O

Lane 18 – 100bp Ladder

Results: Rxn 1 shows amplification with both Bay & Sea Scallop gDNA. The bands are close in size, but look like they would be more distinguishable if run on higher percentage gel and for a longer period of time to get better separation. However, there is contamination in one of the two water samples..

Rxn 2 shows amplification of only the Bay Scallop gDNA.

Rxn 3 shows amplification in both Bay & Sea Scallop gDNA and both bands are of the exact same size.

Rxn 4 shows no amplification in either set of gDNA.

Using the primers used in Rxn 1 will probably allows us to succesfully screen potential hybrids. Just need to remember to use high-percentage agarose gels and run samples for longer periods of time to get sufficient separation.

Used higher annealing temps to improve primer specificity, compared to yesterday’s results. PCR set up and plate layout is here.

See the PCR/plate set up link for samples. Hyperladder is placed between every 12 samples.

Results: See this Google Spreadsheet for a summary of the 4 gels from the last two days.

Because Rony’s results from her PCR did not match her previous results for the positive controls, I ran the PCRs myself on the controls and the hybrids. Anneal temp 50C. PCR set up, samples, etc. are here.

Results: The positive controls still do not match the results Rony got in two consecutive PCR attempts. However, the Bay_Actin_Rv2 and Sea_Actin_Rv3 primers do result in clearly distinguishable differences between bay and sea scallop gDNA. Lane 2 (bay gDNA/bay actin primer) has a large, ~1000bp band and lane 7 (sea gDNA/sea actin primer) produces a ~300bp band. Unfortunately, this does provide us with any useful info. Something needs to be reworked (i.e. possibly new target gene) in order to start getting some useful results.

However, the results we did get definitely confirm that the hybrids are NEITHER hybrids nor bay scallop, due to the fact that no bands are present in any other sample than the bay scallop gDNA samples.