Extracting DNA from Cell and Supernatant Fluid from Myd88 and Dv-1 DiNV Test Infections
On the 13th, the day 5 samples were taken: supernatant was frozen at -80 in 1.5mL tubes and 1mL of 1X PBS was added to each well of the plate and the plate was frozen at -80C as well. I followed the same basic protocol as the last cell/supernatant extraction I did, except I halved all of the volumes.
Sample info:
| sample_ID | extraction_number | plate_day | infection | cell_type | sample_type | date_frozen | PBS_added | date_put_in_tube |
|---|---|---|---|---|---|---|---|---|
| D0A1S | 1 | 0 | yes | Dv1 | supernatant | 20230208 | NA | 20230208 |
| D0A2S | 2 | 0 | yes | Myd88 | supernatant | 20230208 | NA | 20230208 |
| D0A3S | 3 | 0 | no | Dv1 | supernatant | 20230208 | NA | 20230208 |
| D0A4S | 4 | 0 | no | Myd88 | supernatant | 20230208 | NA | 20230208 |
| D0B1S | 5 | 0 | yes | Dv1 | supernatant | 20230208 | NA | 20230208 |
| D0B2S | 6 | 0 | yes | Myd88 | supernatant | 20230208 | NA | 20230208 |
| D0B3S | 7 | 0 | no | Dv1 | supernatant | 20230208 | NA | 20230208 |
| D0B4S | 8 | 0 | no | Myd88 | supernatant | 20230208 | NA | 20230208 |
| D0C1S | 9 | 0 | yes | Dv1 | supernatant | 20230208 | NA | 20230208 |
| D0C2S | 10 | 0 | yes | Myd88 | supernatant | 20230208 | NA | 20230208 |
| D0C3S | 11 | 0 | no | Dv1 | supernatant | 20230208 | NA | 20230208 |
| D0C4S | 12 | 0 | no | Myd88 | supernatant | 20230208 | NA | 20230208 |
| D0A1C | 13 | 0 | yes | Dv1 | cells | 20230208 | 1mL | 20230214 |
| D0A2C | 14 | 0 | yes | Myd88 | cells | 20230208 | 1mL | 20230214 |
| D0A3C | 15 | 0 | no | Dv1 | cells | 20230208 | 1mL | 20230214 |
| D0A4C | 16 | 0 | no | Myd88 | cells | 20230208 | 1mL | 20230214 |
| D0B1C | 17 | 0 | yes | Dv1 | cells | 20230208 | 1mL | 20230214 |
| D0B2C | 18 | 0 | yes | Myd88 | cells | 20230208 | 1mL | 20230214 |
| D0B3C | 19 | 0 | no | Dv1 | cells | 20230208 | 1mL | 20230214 |
| D0B4C | 20 | 0 | no | Myd88 | cells | 20230208 | 1mL | 20230214 |
| D0C1C | 21 | 0 | yes | Dv1 | cells | 20230208 | 1mL | 20230214 |
| D0C2C | 22 | 0 | yes | Myd88 | cells | 20230208 | 1mL | 20230214 |
| D0C3C | 23 | 0 | no | Dv1 | cells | 20230208 | 1mL | 20230214 |
| D0C4C | 24 | 0 | no | Myd88 | cells | 20230208 | 1mL | 20230214 |
| D5A1S | 25 | 5 | yes | Dv1 | supernatant | 20230213 | NA | 20230213 |
| D5A2S | 26 | 5 | yes | Myd88 | supernatant | 20230213 | NA | 20230213 |
| D5A3S | 27 | 5 | no | Dv1 | supernatant | 20230213 | NA | 20230213 |
| D5A4S | 28 | 5 | no | Myd88 | supernatant | 20230213 | NA | 20230213 |
| D5B1S | 29 | 5 | yes | Dv1 | supernatant | 20230213 | NA | 20230213 |
| D5B2S | 30 | 5 | yes | Myd88 | supernatant | 20230213 | NA | 20230213 |
| D5B3S | 31 | 5 | no | Dv1 | supernatant | 20230213 | NA | 20230213 |
| D5B4S | 32 | 5 | no | Myd88 | supernatant | 20230213 | NA | 20230213 |
| D5C1S | 33 | 5 | yes | Dv1 | supernatant | 20230213 | NA | 20230213 |
| D5C2S | 34 | 5 | yes | Myd88 | supernatant | 20230213 | NA | 20230213 |
| D5C3S | 35 | 5 | no | Dv1 | supernatant | 20230213 | NA | 20230213 |
| D5C4S | 36 | 5 | no | Myd88 | supernatant | 20230213 | NA | 20230213 |
| D5A1C | 37 | 5 | yes | Dv1 | cells | 20230213 | 1mL | 20230214 |
| D5A2C | 38 | 5 | yes | Myd88 | cells | 20230213 | 1mL | 20230214 |
| D5A3C | 39 | 5 | no | Dv1 | cells | 20230213 | 1mL | 20230214 |
| D5A4C | 40 | 5 | no | Myd88 | cells | 20230213 | 1mL | 20230214 |
| D5B1C | 41 | 5 | yes | Dv1 | cells | 20230213 | 1mL | 20230214 |
| D5B2C | 42 | 5 | yes | Myd88 | cells | 20230213 | 1mL | 20230214 |
| D5B3C | 43 | 5 | no | Dv1 | cells | 20230213 | 1mL | 20230214 |
| D5B4C | 44 | 5 | no | Myd88 | cells | 20230213 | 1mL | 20230214 |
| D5C1C | 45 | 5 | yes | Dv1 | cells | 20230213 | 1mL | 20230214 |
| D5C2C | 46 | 5 | yes | Myd88 | cells | 20230213 | 1mL | 20230214 |
| D5C3C | 47 | 5 | no | Dv1 | cells | 20230213 | 1mL | 20230214 |
| D5C4C | 48 | 5 | no | Myd88 | cells | 20230213 | 1mL | 20230214 |
DNA Extraction
- All samples were thawed on ice
- Lysis solution was chilled on ice until opaque
- When the plates were thawed, I pipetted the liquid into labeled tubes (see spreadsheet above)
- The cell tubes were then spun down for 5 minutes at 1,500g in the centrifuge to try to pellet the cells (I thought the volume of PBS was too much)
- Then I removed 600ul of liquid from each of the cell tubes, leaving about 300ul (to hopefully concentrate it)
- Made a new 1.5mL tube for each of the 48 samples
- Mixed all samples before pipetting
- Added 50ul of supernatant sample to their designated tubes (1-12 and 25-36)
- Added 1ul of 1000:1 diluted lambda DNA to each supernatant extraction tube
- Added 50ul of cell sample to their designated tubes (13-24 and 37-48)
- Using the Qiagen Puregene Blood and Tissue reagents/method
- Turned on the heat blocks to 65C and 37C and chilled the cell lysis solution on ice until cloudy
- 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 1:100, 2ul in 198ul of nuclease free water
- Let tubes come to room temp after the heat block
- Added 3ul of 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 (had to be done in 2 batches)
- 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 (2 batches)
- 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 (2 batches)
- 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