Kaitlyn’s notebook: primers on known geoduck hemolymph samples

Previous qPCR test on pooled samples determined these 4 primers would be run on known samples. Ran qPCR as previously described.

Results from qPCR on known samples:

Two runs had to be done to be able to include all samples.

• • Run 1 Bio-Rad file and Run 2 Bio-Rad file
  • Run 1 .csv file and Run 2 .csv file

Results

• • contamination in no template controls (NTC)
    • unknown when contamination could have occurred
      • Water used for NTC seems unlikely since similar amplification is occurring in each sample
      • Possible contamination in primers, master mix, or DNase
        • Tris-EDTA added to lyophilized primers?
          • used 2017 DNase for RT

 

Regardless, there is non-specific amplification which also occurred in the last qPCR run on pooled samples.

The non-specific binding could instead be the presence of leftover DNA from a less effective DNase (since it was from 2017). In order to test this:

• • one RNA sample left (G38) that hasn’t been DNased
    • run qPCR on 4ng RNA from G38 (equivalent to RNA amount run in previous qPCRs)
      • Include NTC to test if primers are contaminated as well

I can dilute my cDNA 1:10 to try to make it last longer which can be seen in amplification image. I used up a substantial portion testing the 4 selected primers during these runs.

Next, Sam suggested I run pooled samples at a gradient of temperatures to identify an ideal temperature with reduced non-specific binding.