Extracting DNA from Day0 CCM and DiNV, and Dead from DiNV Infection Flies to Use for an Experiment to Determine Best DNA Concentration for qPCR from Fly Infections

For this there were certain flies we wanted to use, and their DNA needed to be extracted first.

2 flies from a day 0 CCM treatment. Samples 7 and 9 from the second replicate female and male DiNV injection experiment. Infection notebook here, sample info here. Both samples are male.

2 flies from a day 0 DiNV treatment. Samples 16 and 19 from the second replicate female and male DiNV injection experiment. Infection notebook here, sample info here. Both samples are male.

2 flies dead from DiNV infection. Samples 29 and 42 from the first replicate female and male DiNV injection experiment. Infection notebook here. sample info here. Both samples are male.

DNA extraction followed this protocol exactly. Samples sat on the bench for ~3 hours before Qubit, dilution, and freezing at -20 instead of overnight. Care was taken to separate out the control and infected samples, and 2 extraction controls were done at the same time.

Afterwards a broad range DNA Qubit, following this protocol exactly was performed to determine DNA concentrations.

sample DNA concentration
7 11.4ng/ul
9 12.3ng/ul
16 11.5ng/ul
19 12.1ng/ul
29 15ng/ul
42 8.92ng/ul

Rob and I had decided that we would like to use 1ng and 0.5ng as input amounts of DNA into the qPCR, so the samples had to be diluted.

Based off of the Qubit values, the samples were diluted to 1ng/ul and 0.5ng/ul with the volumes in the table below in strip tubes:

sample volume stock DNA for 1ng/ul volume DNA hydration solution for 1ng/ul volume 1ng/ul DNA for 0.5ul volume DNA hydration solution for 0.5ng/ul
7 3ul 31.2ul 5ul 5ul
9 3ul 33.9ul 5ul 5ul
16 3ul 31.5ul 5ul 5ul
19 3ul 33.3ul 5ul 5ul
29 3ul 42ul 5ul 5ul
42 3ul 23.6ul 5ul 5ul

For these, all samples were kept on ice and vortexed and spun down before use. All DNA was frozen at -20C.