Extracting DNA and PCRs for CO1, TPI, 115, and p47 on Single Flies from the Test Infections for Sterility

All samples are from the test infections started on 7/34. Sample info can be found here.

20230731 DNA extractions

  • Flies were frozen in the morning (see above notebook for methods), and immediately extracted with day 0 flies
  • Order was not randomized because all flies were extracted at once
  • An extraction control was used for balance reasons and to test extraction method, this was a tube with no fly that went through all the same steps
  • DNA was extracted from each fly using the general single fly extraction protocol, with a few changes
    • Notably, filter tips were used in all steps, and instead of using the motorized pestle grinder to homogenize flies, the pestles were used to homogenize the flies by hand to avoid any splashing of fly fluid.
    • Additionally all supernatants were pipetted out of tubes instead of poured

20230803 30 Cycle PCRs

PCRs on CO1, TPI, p47, and 115 were done accordingly to the general PCR protocol with the primers, and their information can be found here. The p47 and 115 samples were amplified in 30 cycles. CO1 and TPI primers were used in the same tube, while the p47 and 115 primers were used in separate reaction tubes, but with the same program. Each primer set had a negative control and a positive control (has fly DNA and virus DNA). The extraction control was used in all PCRs as a check of the extraction process.

After the PCRs, a 2% gel was run in the largest gel box: 3.3g agarose, 165mL 1X TAE, and 2ul of Midori stain. The gel was ran for 45 minutes at 90V. Because there are 44 wells needed per primer, 2 large gels were run.

CO1 and TPI gel:

30 cycle p47 and 115 gel:

20230804 35 Cycle PCRs

It could be that some of these samples are contaminated, but it doesn’t show up in the 30 cycle PCRs. So I decided to do all the test infection samples with 35 cycles with 115 and p47, as well as the stock innubila (see here).

p47, and 115 were done accordingly to the general PCR protocol with the primers, and their information can be found here. The p47 and 115 primers were used in separate reaction tubes, but in the same program. Each primer set had a negative control and a positive control (has fly DNA and virus DNA). The extraction control was used in all PCRs as a check of the extraction process.

After the PCRs, a 2% gel was run in the largest gel box: 3.3g agarose, 165mL 1X TAE, and 2ul of Midori stain. The gel was ran for 45 minutes at 90V. Because there were too many samples, 2 large gels were run.

Stock D. innubila 115 35 cycles:

Stock D. innubila p47 35 cycles:

Test infections p47 and 115 35 cycles:

Looks like for all of these the sterile poked flies or stock flies are virus negative. The only thing that worries me is the extraction control for the test infections which is positive for 35 cycles.