Extracting DNA from Dv-1 and Myd88 Cells and Supernatant from the DiNV Infection Experiment
For flask samples, there is very little liquid in them. I added 1mL of 1X PBS to each flask to make it a usable sample. I also did a Lambda DNA spike for the supernatant samples because they likely contain low amounts of DNA. I also decided to do an extraction replicate for each sample because there was so much sample.
I also did a modified extraction procedure based off of Kent’s viral DNA extraction protocol and the single fly extraction protocol. Basically I doubled Kent’s volumes and added in heated incubation steps.
The sampling datasheet looks like this:
| Sample_ID | Cell_type | Day | infection_status | type | flask_replicate | extraction_replicate | lambda_spike | PBS_added |
|---|---|---|---|---|---|---|---|---|
| 1 | Dv-1 | 0 | yes | supernatant | 1 | yes | NA | |
| 1.1 | Dv-1 | 0 | yes | supernatant | yes | yes | NA | |
| 2 | Dv-1 | 0 | yes | cells | 1 | NA | 1mL | |
| 2.1 | Dv-1 | 0 | yes | cells | yes | NA | 1mL | |
| 3 | Myd88 | 0 | yes | supernatant | 1 | yes | NA | |
| 3.1 | Myd88 | 0 | yes | supernatant | yes | yes | NA | |
| 4 | Myd88 | 0 | yes | cells | 1 | NA | 1mL | |
| 4.1 | Myd88 | 0 | yes | cells | yes | NA | 1mL | |
| 5 | Dv-1 | 5 | yes | supernatant | 1 | yes | NA | |
| 5.1 | Dv-1 | 5 | yes | supernatant | yes | yes | NA | |
| 6 | Dv-1 | 5 | yes | cells | 1 | NA | 1mL | |
| 6.1 | Dv-1 | 5 | yes | cells | yes | NA | 1mL | |
| 7 | Dv-1 | 5 | yes | supernatant | 2 | yes | NA | |
| 7.1 | Dv-1 | 5 | yes | supernatant | yes | yes | NA | |
| 8 | Dv-1 | 5 | yes | cells | 2 | NA | 1mL | |
| 8.1 | Dv-1 | 5 | yes | cells | yes | NA | 1mL | |
| 9 | Dv-1 | 5 | no | supernatant | 1 | yes | NA | |
| 9.1 | Dv-1 | 5 | no | supernatant | yes | yes | NA | |
| 10 | Dv-1 | 5 | no | cells | 1 | NA | 1mL | |
| 10.1 | Dv-1 | 5 | no | cells | yes | NA | 1mL | |
| 11 | Myd88 | 5 | yes | supernatant | 1 | yes | NA | |
| 11.1 | Myd88 | 5 | yes | supernatant | yes | yes | NA | |
| 12 | Myd88 | 5 | yes | cells | 1 | NA | 1mL | |
| 12.1 | Myd88 | 5 | yes | cells | yes | NA | 1mL | |
| 13 | Myd88 | 5 | yes | supernatant | 2 | yes | NA | |
| 13.1 | Myd88 | 5 | yes | supernatant | yes | yes | NA | |
| 14 | Myd88 | 5 | yes | cells | 2 | NA | 1mL | |
| 14.1 | Myd88 | 5 | yes | cells | yes | NA | 1mL | |
| 15 | Myd88 | 5 | no | supernatant | 1 | yes | NA | |
| 15.1 | Myd88 | 5 | no | supernatant | yes | yes | NA | |
| 16 | Myd88 | 5 | no | cells | 1 | NA | 1mL | |
| 16.1 | Myd88 | 5 | no | cells | yes | NA | 1mL |
Preparing Samples
- All supernatant tubes and flasks were thawed on ice
- The stock Lambda DNA (from Kent) was thawed on ice
- Lambda DNA was vortexed and spun down
- A dilution of 1:1000 was done for the Lambda: 1ul in 1mL of nuclease fre water. This was vortexed, spun down, and kept on ice
- Once thawed, each flask had 1mL of 1X PBS added to it, swished back and forth, and sucked up and added to a 1.5mL tube for easy access
- I also made 1mL aliquots of supernatant fluid for each sample in 1.5mL tubes for easy access
- For each sample, I made 2 tubes for extraction. They were labeled 1-16.1 as in the above table
- For each sample, I pipetted 100ul of sample into 2 tubes each (one is the extraction replicate)
- Note here that potentially I was not consistent with pipetting a similar sample to each replicate tube
- For all the supernatant samples and their extraction replicates, I needed to add Lambda DNA as an extraction control. Kent says he uses 1ul of 1:1000 diluted DNA, but because I decided to double his volumes I used 2ul
- Added 2ul of diluted Lambda DNA to tubes 1, 1.1, 3, 3.1, 5, 5.1, 7, 7.1, 9, 9.1, 11, 11.1, 13, 13.1, 15, and 15.1
DNA Extraction
- 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 500ul 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 6ul 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 200ul 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 600ul of fresh 100% ethanol with final tube labels
- Transferred the supernatant from the centrifugation (~800ul) 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 600ul 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
Qubit of extracted samples 20230125
- I have 32 samples, plus 2 standards, plus 3 for error
- N= 37
- 37 * 199 = 7,363ul buffer
- 37ul Quant IT reagent
- Mixed quantification buffer
- Prepared sample and standard Qubit tubes
- Added 190ul Quant buffer to the standard tubes
- Added 199ul Quant buffer to the sample tubes
- Added 10ul standards to the standard tubes
- Added 1ul sample to the sample tubes
- Vortexed and spun down tubes
- Let tubes sit ~2 min
- Quantified samples:
| Sample_ID | Cell_type | Day | infection_status | type | flask_replicate | extraction_replicate | lambda_spike | PBS_added | extraction_date | qubit_quantity |
|---|---|---|---|---|---|---|---|---|---|---|
| 1 | Dv-1 | 0 | yes | supernatant | 1 | yes | NA | 20230124 | too low | |
| 1.1 | Dv-1 | 0 | yes | supernatant | yes | yes | NA | 20230124 | 2.67 | |
| 2 | Dv-1 | 0 | yes | cells | 1 | NA | 1mL | 20230124 | too low | |
| 2.1 | Dv-1 | 0 | yes | cells | yes | NA | 1mL | 20230124 | too low | |
| 3 | Myd88 | 0 | yes | supernatant | 1 | yes | NA | 20230124 | too low | |
| 3.1 | Myd88 | 0 | yes | supernatant | yes | yes | NA | 20230124 | too low | |
| 4 | Myd88 | 0 | yes | cells | 1 | NA | 1mL | 20230124 | 5.16 | |
| 4.1 | Myd88 | 0 | yes | cells | yes | NA | 1mL | 20230124 | 4.18 | |
| 5 | Dv-1 | 5 | yes | supernatant | 1 | yes | NA | 20230124 | too low | |
| 5.1 | Dv-1 | 5 | yes | supernatant | yes | yes | NA | 20230124 | 5.05 | |
| 6 | Dv-1 | 5 | yes | cells | 1 | NA | 1mL | 20230124 | 48.1 | |
| 6.1 | Dv-1 | 5 | yes | cells | yes | NA | 1mL | 20230124 | 7.3 | |
| 7 | Dv-1 | 5 | yes | supernatant | 2 | yes | NA | 20230124 | 13.2 | |
| 7.1 | Dv-1 | 5 | yes | supernatant | yes | yes | NA | 20230124 | 13.7 | |
| 8 | Dv-1 | 5 | yes | cells | 2 | NA | 1mL | 20230124 | 44.3 | |
| 8.1 | Dv-1 | 5 | yes | cells | yes | NA | 1mL | 20230124 | 12.9 | |
| 9 | Dv-1 | 5 | no | supernatant | 1 | yes | NA | 20230124 | 10.6 | |
| 9.1 | Dv-1 | 5 | no | supernatant | yes | yes | NA | 20230124 | 12.1 | |
| 10 | Dv-1 | 5 | no | cells | 1 | NA | 1mL | 20230124 | 45.6 | |
| 10.1 | Dv-1 | 5 | no | cells | yes | NA | 1mL | 20230124 | 16.9 | |
| 11 | Myd88 | 5 | yes | supernatant | 1 | yes | NA | 20230124 | 11.2 | |
| 11.1 | Myd88 | 5 | yes | supernatant | yes | yes | NA | 20230124 | 11.5 | |
| 12 | Myd88 | 5 | yes | cells | 1 | NA | 1mL | 20230124 | 20.1 | |
| 12.1 | Myd88 | 5 | yes | cells | yes | NA | 1mL | 20230124 | 20.1 | |
| 13 | Myd88 | 5 | yes | supernatant | 2 | yes | NA | 20230124 | 14.5 | |
| 13.1 | Myd88 | 5 | yes | supernatant | yes | yes | NA | 20230124 | 14.2 | |
| 14 | Myd88 | 5 | yes | cells | 2 | NA | 1mL | 20230124 | 18.3 | |
| 14.1 | Myd88 | 5 | yes | cells | yes | NA | 1mL | 20230124 | 16.4 | |
| 15 | Myd88 | 5 | no | supernatant | 1 | yes | NA | 20230124 | 14.2 | |
| 15.1 | Myd88 | 5 | no | supernatant | yes | yes | NA | 20230124 | 12.8 | |
| 16 | Myd88 | 5 | no | cells | 1 | NA | 1mL | 20230124 | 14 | |
| 16.1 | Myd88 | 5 | no | cells | yes | NA | 1mL | 20230124 | 13.9 |
Some samples are too low, but could be just below the detection threshold of 2ng/ul. There is also considerable variability in extraction output for some replicates, while others are very consistent. It could be that the samples I gave the replicate had settled and had less cells or sample when I thought I was giving them the same. Or I lost pellets places.
Samples were frozen at -20 after this.