Amplifying rp49 (control product) and p47 (DiNV specific) PCR Products on Myd88 and W1118 Flies Infected with DiNV

Info on fly infections here

Info on how 12 flies were randomly chosen and DNA extracted here. That link also include the table that indicates which sample is which extraction number.

20220517 rp49 PCR

  • Primers are #s 8 and 9 in the oligo database
  • Database also says they amplify a band a ~200bp when using a 55 degree C annealing temp
  • I diluted the primers to 10uM by using 10ul of primer in 90ul of molecular grade water
  • I have 12 samples, I need a negative control, and I have some W1118 DNA from Emma as a positive control
  • Made mastermix on ice:
    • 5ul GoTaq * 15 = 75ul
    • 0.25ul rp49-F * 15 = 3.75ul
    • 0.25ul rp49-R * 15 = 3.75ul
    • 3.5ul molec grade water * 15 = 52.5ul
  • Mix was vortexed and spun down
  • Added 9ul of master mix to each strip tube
  • Added 1ul of DNA to each tube and 1ul of water to the control tubes
  • Tubes were ordered by extraction #
  • Placed in the 55TEST PCR program
  • Afterwards, the samples were run on a 2% gel for 35 minutes at 90V

This looks good! All the samples except 7 amplified for the melanogaster gene. Sample 7 had too low to read Qubit from the extraction, so there may be almost no DNA in there.

20220519 p47_q PCR

  • Using the QPCR version of the p47 primers, this makes a ~100bp product
  • I used a puregene kit DNA extracted from DV-1 cells infected with DiNV as my positive control
  • Made mastermix on ice:
    • 5ul GoTaq * 15 = 75ul
    • 0.25ul p47_q-F * 15 = 3.75ul
    • 0.25ul p47_q-R * 15 = 3.75ul
    • 3.5ul molec grade water * 15 = 52.5ul
  • Mix was vortexed and spun down
  • Added 9ul of master mix to each strip tube
  • Added 1ul of DNA to each tube and 1ul of water to the control tubes
  • Tubes were ordered by extraction #
  • Placed in the 55TEST PCR program
  • Afterwards, the samples were run on a 2% gel for 35 minutes at 90V by Emma the next day

No amplification except for in the positive control. This is somewhat to be expected, I may not have poked the flies hard enough or DiNV wasn’t able to infect them well. I should have tried amplifying the liquid I infected them with. I will try that next.