Extracting DNA from Ultrafiltered Viral Fluids
To do qPCR on the concentrated, non-concentrated, flow through, and wash samples from the test viral fluid concentration I need to have DNA extracted. I used the 50ul aliquots from those samples, and left the others frozen. See sample list below. I also extracted DNA from 1 40ul aliquot of D. innubila supernatant that I got from Kent that is “day 26” from way earlier in the experiment (not listed on the table).
I followed the same extraction procedure as any other cell culture supernatant sample (like this one).
| sample_ID | date_collected | date_frozen | number_freeze_thaws | sample_type | volume_ul | concentration volume | concentration_time | concentration_tube |
|---|---|---|---|---|---|---|---|---|
| 1 | 20230222 | 20230222 | non-concentrated | 50 | NA | NA | NA | |
| 4 | 20230222 | 20230222 | 1 | concentrated | 50 | 4mL | 20 min | sartorius 10,000 MWC |
| 6 | 20230222 | 20230222 | 1 | flow-through | 50 | 4mL | 20 min | sartorius 10,000 MWC |
| 8 | 20230213 | 20230213 | 2 | non-concentrated | 50 | NA | NA | NA |
| 11 | 20230213 | 20230222 | 1 | concentrated | 50 | 4mL | 20 min | sartorius 10,000 MWC |
| 13 | 20230213 | 20230222 | 1 | flow-through | 50 | 4mL | 20 min | sartorius 10,000 MWC |
| 17 | 20230222 | 20230222 | wash | 50 | 4mL | 20 min | sartorius 10,000 MWC | |
| 18 | 20230213 | 20230222 | 1 | wash | 50 | 4mL | 20 min | sartorius 10,000 MWC |
- All samples were thawed on ice
- Lysis solution was chilled on ice until opaque
- Added 1ul of 1000:1 diluted lambda DNA to each sample tube
- Using the Qiagen Puregene Blood and Tissue reagents/method
- Turned on the heat blocks to 65C and 37C
- Added 250ul of chilled cell lysis solution to each tube and pipette mixed 10X
- Incubated tubes in the heat block at 65C for 10 minutes
- Diluted RNase A. We have a 10mg/mL stock now, and I did some looking around in our protocols and it seems like we want 2mg/mL per 150ul of liquid. So because I am using 300ul of liquid here, I wanted to use 4mg/mL per sample. I am still not sure if I was doing this calculation correctly
- 4ul RNase A 10mg/mL
- 6ul nuclease free water
- Pipette mixed
- Let tubes come to room temp after the heat block
- Added 1ul of the diluted RNase A to each tube
- Flipped the tubes 25X to mix them
- Incubated tubes at 37C for 30 minutes in the heat block
- Let tubes come to room temp after incubation
- Added 100ul of protein precipitation solution to each tube
- Vortexed tubes ~10 sec
- Placed tubes on ice for 5 min
- Centrifuged tubes 16,300g for 3 minutes
- Made new tubes with 300ul of fresh 100% isopropanol with final tube labels
- Transferred the supernatant from the centrifugation (~400ul) to the new tubes with the isopropanol
- Inverted the tubes 50X
- Centrifuged the tubes 16,300g for 5 minutes
- Discarded the supernatant
- Note that I didn’t see a pellet in any of the samples
- Added 300ul of fresh 70% ethanol to each tube
- Inverted 3X
- Centrifuged tubes 16,300g 5 min
- Discarded supernatant
- Let tubes air dry upside down on a kim wipe for ~1 hour
- Resuspended DNA in 20ul of DNA hydration solution and let tubes sit overnight
- Tubes were put in the -20 the next day