Montipora Larvae DNA/RNA Extraction 4
Using Larvae Extraction Protocol on 6 of Ariana’s Montipora Coral Larvae Stored in DNA/RNA Shield
Goal: Use optimized extraction method on 6 samples for Ariana’s project and elute the three samples above D29 in only 70ul of ultrapure water instead of 100ul
Results: Good DNA and RNA quantity and quality, RNA purity is ok
Take aways: I made new wash buffer for this extraction and the purity is noticeably better than the last extraction. However the wash buffers should be exactly the same, it could have been the samples. The RNA purity is still not completely good.
| Sample | Timepoint | TP | Volume Shield in Storage | # Larvae |
|---|---|---|---|---|
| D2 | 1hpf | TP1 | 1mL | 250ul eggs |
| D13 | 65hpf | TP4 | 500ulmL | 50ul |
| D24 | 163hpf | TP6 | 500ul | 50ul |
| D29 | 183hpf | TP9 | 300ul | 15-20 indiviuals |
| D37 | 231hpf | TP10 | 300ul | 15-20 individuals |
| D40 | 231hpf | TP10 | 300ul | 15-20 individuals |
Based off of the sample info and how many larvae were present in each tube, save fractions were made for samples D2, D13, and D24, but not D29, D37, and D40.
| Sample | Volume Shield Added at Extraction | Save Fraction? | Saved Volume |
|---|---|---|---|
| D2 | 0 | yes | 500ul |
| D13 | 500ul | yes | 500ul |
| D24 | 500ul | yes | 500ul |
| D29 | 200ul | no | - |
| D37 | 200ul | no | - |
| D40 | 200ul | no | - |
I eluted the RNA of D29, D37 and D40 in 70ul of ultrapure water instead of 100ul
Extraction
- Extraction protocol followed exactly
- Extraction proceeded with 500ul of each sample
- 500ul of DNA/RNA lysis buffer was used for each sample
- 1mL of 100% ethanol was used for each sample
- Samples D29, D37 and D40 were eluted with 40ul ultrapure water in the first elution and 30ul in the second
QC
Qubit
- Broad Range dsDNA and Borad Range RNA Qubit protocol
- All samples were read twice
BR DNA:
| Sample | Standard 1 | Standard 2 | DNA 1 ng/ul | DNA 2 ng/ul | Average ng/ul |
|---|---|---|---|---|---|
| D2 | 172 | 18281 | too low | - | - |
| D13 | - | - | 31.6 | 31.4 | 31.5 |
| D24 | - | - | 18.8 | 18.7 | 18.75 |
| D29 | - | - | 14.3 | 14.1 | 14.2 |
| D37 | - | - | 23 | 23 | 23 |
| D40 | - | - | 12.1 | 12 | 12.05 |
BR RNA:
| Sample | Standard 1 | Standard 2 | RNA 1 ng/ul | RNA 2 ng/ul | Average ng/ul |
|---|---|---|---|---|---|
| D2 | 375 | 7873 | 250 | 250 | 250 |
| D13 | - | - | 94.8 | 94.6 | 94.7 |
| D24 | - | - | 32 | 32.2 | 32.1 |
| D29 | - | - | 28.4 | 28.2 | 28.3 |
| D37 | - | - | 36 | 36.2 | 36.1 |
| D40 | - | - | 17.6 | 17.6 | 17.6 |
NanoDrop
- Followed NanoDrop RNA Protocol
| Sample | 260/230 | 260/280 |
|---|---|---|
| D3 | 2.13 | 2.12 |
| D13 | 1.65 | 2.03 |
| D24 | 1.14 | 2.06 |
| D29 | 1.10 | 1.89 |
| D37 | 1.03 | 1.92 |
| D40 | 1.08 | 1.87 |
Full Results:
Traces:
Gel
- 1% gel run for 1 hour at 100V
- Gel Protocol
TapeStation
- Followed RNA TapeStation protocol
| Sample | RIN score |
|---|---|
| D2 | 8.1 |
| D13 | 8.9 |
| D24 | 8.7 |
| D29 | 9.4 |
| D37 | 9.3 |
| D40 | 9.0 |