Excluding the no template controls (NTC), all samples produced amplification. Will require DNasing before making cDNA.
Related to the qPCR I ran earlier today with these same primers, the efficiencies of the reactions on this plate are significantly better (i.e. normal; >80% efficiencies) than the earlier qPCR. The improved efficiency would also explain why the positive control comes up two cycles earlier on this run.
In the amplification plots below, the positive control reps are the two lines coming up at cycle ~20.
Ran a qPCR on all cDNA samples from the V.vulnificus exposure experiment from 20110111. This qPCR was to test 2 of 4 potential normalizing genes to evaluate which genes show the least amount of effect from the treatments in this experiment. Primers for actin used were Cg_Actin_306_F (SR ID: 1170), Cg_Actin_408_R (SR ID: 1171). Samples were run in duplicate. Master mix calcs are here. The master mix info is the same that was used earlier today, but with the primers noted above, not those listed on the calcs page. Plate layout, cycling params, etc., can be seen in the qPCR Report (see Results).
Actin: Average Cq = 20.21, Standard Deviation = 1.22
GAPDH: Average Cq = 24.42, Standard Deviation = 0.519
Based on the results from the 4 normalizing genes examined, I will use GAPDH as the normalizing gene due to it having the lowest standard deviation of the 4 normalizing genes. Will perform another qPCR to run a duplicate of GAPDH so that we have a second rep.
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.