Puregene Single Fly DNA Extractions: 12 Myd88 and W1118 Flies from Infections with DiNV
- Using Qiagen Puregene kit
- Randomized range of fly sample spreadsheet and gave each sample a shorter name
- Selected the first 12 to do today, as to not do too many because I haven’t done this extraction before
| Sample | Sex | Day | Date_infected | Date_frozen | Extraction_number |
|---|---|---|---|---|---|
| W1118_m_d6_5 | M | 6 | 20220502 | 20220508 | 1 |
| Md88_f_d3_4 | F | 3 | 20220502 | 20220505 | 2 |
| W1118_m_d3_6 | M | 3 | 20220502 | 20220505 | 3 |
| W1118_f_d6_4 | F | 6 | 20220502 | 20220508 | 4 |
| Myd88_f_d6_10 | F | 6 | 20220502 | 20220508 | 5 |
| Myd88_f_d1_3 | F | 1 | 20220502 | 20220502 | 6 |
| W1118_m_d6_3 | M | 6 | 20220502 | 20220508 | 7 |
| Myd88_m_d6_7 | M | 6 | 20220502 | 20220508 | 8 |
| Myd88_f_d6_2 | F | 6 | 20220502 | 20220508 | 9 |
| Myd88_m_d3_4 | M | 3 | 20220502 | 20220505 | 10 |
| Myd88_f_d1_1 | F | 1 | 20220502 | 20220502 | 11 |
| W1118_m_d3_3 | M | 3 | 20220502 | 20220505 | 12 |
- Made aliquots of lysis solution, protein precipitation solution, and hydration solution
- Chilled cell lysis solution on ice
- Added 100ul cold cell lysis solution to each fly tube
- Homogenized each fly with a motorized pestle for ~5 seconds. Each fly got a new pestle. Pestles had been autoclaved
- Spun down tubes
- Incubated tubes in the heat block for 15 minutes at 65 degrees C
- Made a dilution of RNase A from 100mg/mL to 1mg/mL by doing a 1:100 dilution, 1ul in 99ul molecular grade water
- After incubation, let tubes cool to room temp
- Added 2ul of the diluted RNase A
- Inverted tubes to mix 25 times
- Spun down tubes briefly
- Incubated tubes at 37 degrees C for 40 minutes
- Cooled tubes to room temperature
- Added 33ul of protein precipitation solution to each tube
- Vortexed tubes for 10 seconds each
- Placed tubes on ice for 5 minutes
- Centrifuged tubes at ~14,000rpm for 3 minutes
- Made final 1.5mL tubes with 100ul of 100% isopropanol in each
- After centrifugation, transferred the supernatant to the isopropanol tubes
- Inverted tubes gently 50 times to mix
- Centrifuged tubes ~14,000rpm for 5 minutes
- Afterwards all tubes had a pellet visible
- Discarded supernatant
- Added 100ul freshly prepared 70% ethanol to each tube
- Inverted tubes ~3 times to mix
- Centrifuged tubes ~14,000rpm 1 minute
- Tubes 3 and 7 no longer seemed to have a pellet
- Discarded supernatant
- Let tubes dry on bench on kim wipe for ~1 hour 30 minutes
- Resuspended pellets in 20ul DNA hydration solution
- Let DNA resuspend overnight on the benchtop
20220512 Qubit
- Using BR dsDNA Qubit
- Made mix:
- 2,985ul buffer
- 15ul reagent
- 190ul mix in standard tubes
- 199ul mix in sample tubes
- 10ul of standard into the standard tubes
- 1ul DNA into the sample tubes
- Vortex, spin down, and incubate in dark for 2 minutes before reading:
| Sample | Extraction_number | Extraction_quant |
|---|---|---|
| W1118_m_d6_5 | 1 | 1ng/ul |
| Md88_f_d3_4 | 2 | 20ng/ul |
| W1118_m_d3_6 | 3 | 2.02ng/ul |
| W1118_f_d6_4 | 4 | 11.1ng/ul |
| Myd88_f_d6_10 | 5 | 21.7ng/ul |
| Myd88_f_d1_3 | 6 | 21.9ng/ul |
| W1118_m_d6_3 | 7 | 1ng/ul |
| Myd88_m_d6_7 | 8 | 12ng/ul |
| Myd88_f_d6_2 | 9 | 18.1ng/ul |
| Myd88_m_d3_4 | 10 | 12.5ng/ul |
| Myd88_f_d1_1 | 11 | 18.1ng/ul |
| W1118_m_d3_3 | 12 | 9.92ng/ul |
** samples that say 1ng/ul had too low to read on the Qubit, but because the BR kit cannot read below 2ng/ul, it could still have DNA in there.