Poc TPC Transect ID via mtORF
Pocillopora species ID for POC ID TPC relative abundance
Equipment and Reagents
- Foward primer FatP6.1 200µM Stock IDT)
- Reverse primer RORF 200µM Stock IDT
- Master Mix EmeraldAmp GT PCR Master Mix
- EmeraldAmp GT PCR Master Mix is a loading-dye-added version of EmeraldAmp MAX PCR Master Mix that is optimized for great performance and convenience in both standard and high-throughput PCR applications.
- Gel Stain Biotium GelGreen Nucleic Acid Gel Stain, 10,000X in Water Fisher Cat NC9728313
- Gel Ladder Zymo 5006-50 1kb ladder ready to load
Apmlification of Pocillopora samples for species ID with mtORF
Samples
| Sample ID | Project | Collection Date |
|---|---|---|
| POC-200 | Moorea TPC Relative Abundance | May 2022 |
| POC-204 | Moorea TPC Relative Abundance | May 2022 |
| POC-216 | Moorea TPC Relative Abundance | May 2022 |
| POC-217 | Moorea TPC Relative Abundance | May 2022 |
| POC-223 | Moorea TPC Relative Abundance | May 2022 |
| POC-228 | Moorea TPC Relative Abundance | May 2022 |
| POC-230 | Moorea TPC Relative Abundance | May 2022 |
| POC-246 | Moorea TPC Relative Abundance | May 2022 |
| POC-278 | Moorea TPC Relative Abundance | May 2022 |
| POC-279 | Moorea TPC Relative Abundance | May 2022 |
| POC-281 | Moorea TPC Relative Abundance | May 2022 |
| POC-285 | Moorea TPC Relative Abundance | May 2022 |
| POC-287 | Moorea TPC Relative Abundance | May 2022 |
| POC-289 | Moorea TPC Relative Abundance | May 2022 |
| POC-292 | Moorea TPC Relative Abundance | May 2022 |
| POC-294 | Moorea TPC Relative Abundance | May 2022 |
| POC-296 | Moorea TPC Relative Abundance | May 2022 |
| POC-257 | Moorea TPC Relative Abundance | May 2022 |
| POC-262 | Moorea TPC Relative Abundance | May 2022 |
| POC-263 | Moorea TPC Relative Abundance | May 2022 |
| POC-264 | Moorea TPC Relative Abundance | May 2022 |
| POC-265 | Moorea TPC Relative Abundance | May 2022 |
| POC-268 | Moorea TPC Relative Abundance | May 2022 |
| POC-306 | Moorea TPC Relative Abundance | May 2022 |
| POC-309 | Moorea TPC Relative Abundance | May 2022 |
| POC-311 | Moorea TPC Relative Abundance | May 2022 |
| POC-322 | Moorea TPC Relative Abundance | May 2022 |
| POC-329 | Moorea TPC Relative Abundance | May 2022 |
| POC-355 | Moorea TPC Relative Abundance | May 2022 |
| POC-356 | Moorea TPC Relative Abundance | May 2022 |
| POC-359 | Moorea TPC Relative Abundance | May 2022 |
| POC-360 | Moorea TPC Relative Abundance | May 2022 |
| POC-362 | Moorea TPC Relative Abundance | May 2022 |
| POC-363 | Moorea TPC Relative Abundance | May 2022 |
| POC-364 | Moorea TPC Relative Abundance | May 2022 |
| POC-365 | Moorea TPC Relative Abundance | May 2022 |
| POC-366 | Moorea TPC Relative Abundance | May 2022 |
| POC-367 | Moorea TPC Relative Abundance | May 2022 |
| POC-368 | Moorea TPC Relative Abundance | May 2022 |
| POC-369 | Moorea TPC Relative Abundance | May 2022 |
| POC-370 | Moorea TPC Relative Abundance | May 2022 |
| POC-372 | Moorea TPC Relative Abundance | May 2022 |
| POC-374 | Moorea TPC Relative Abundance | May 2022 |
| POC-379 | Moorea TPC Relative Abundance | May 2022 |
| POC-382 | Moorea TPC Relative Abundance | May 2022 |
| POC-383 | Moorea TPC Relative Abundance | May 2022 |
| POC-385 | Moorea TPC Relative Abundance | May 2022 |
| POC-386 | Moorea TPC Relative Abundance | May 2022 |
| POC-388 | Moorea TPC Relative Abundance | May 2022 |
| POC-392 | Moorea TPC Relative Abundance | May 2022 |
| POC-393 | Moorea TPC Relative Abundance | May 2022 |
| POC-399 | Moorea TPC Relative Abundance | May 2022 |
| POC-400 | Moorea TPC Relative Abundance | May 2022 |
| POC-401 | Moorea TPC Relative Abundance | May 2022 |
| POC-402 | Moorea TPC Relative Abundance | May 2022 |
| POC-406 | Moorea TPC Relative Abundance | May 2022 |
Positive control = POC Spawning 198 Negative control = H20
PCR
Prepare Reagents
Primers - Flot et al. 2008
- Forward primer FatP6.1 (Added 50µl of stock 2mg/ml plus 950µl of Nuclease Free water and made 3 aliquots of ~320µl each and stored in reagent box at -20)
- FatP6.1 (5′-TTTGGGSATTCGTTTAGCAG-3′)
- Reverse primer RORF (Added 50µl of stock 2mg/ml plus 950µl of Nuclease Free water and made 3 aliquots of ~320µl each and stored in reagent box at -20)
- RORF (5′-SCCAATATGTTAAACASCATGTCA-3′)
- Master Mix EmeraldAmp GT PCR Master Mix
EmeraldAmp GT PCR Master Mix is a loading-dye-added version of EmeraldAmp MAX PCR Master Mix that is optimized for great performance and convenience in both standard and high-throughput PCR applications.
EmeraldAmp GT PCR Master Mix (2x)
Master Mix: For 58 rxns +2 = 60
| Reagent | 1Rxn µl | 32 Rxn µl |
|---|---|---|
| EmeraldAmp GT Mix (2x) | 12.5 | 750 |
| F primer FatP6.1 (10µM) | 0.3 | 18 |
| R primer RORF (10µM) | 0.3 | 18 |
| DNA | 1 | NA |
| H2O | 10.9 | 654 |
| Total volume | 25 | 1500 |
Thermal Cycling Conditions
- [94°C 60 secondes] 1 cycle
- [94°C 30 sec,53°C 30 sec, 72°C 75 sec] 30 cycles
- [72°C 5 minutes] 1 cycle
- [4°C infinity]
PCR Gel protocol
prepared two 100ml gel using 50ml of 1xTAE + 1.5g Agarose, melted gel for ~120 sec in the microwave and let cool and added 1µl Gelgreen and let harden. Ran gel at 100V for 45 minutes in 1x TAE.
Gel Samples
Gel 1
Top Row
| Sample ID | Project | Collection Date |
|---|---|---|
| POC-200 | Moorea TPC Relative Abundance | May 2022 |
| POC-204 | Moorea TPC Relative Abundance | May 2022 |
| POC-216 | Moorea TPC Relative Abundance | May 2022 |
| POC-217 | Moorea TPC Relative Abundance | May 2022 |
| POC-223 | Moorea TPC Relative Abundance | May 2022 |
| POC-228 | Moorea TPC Relative Abundance | May 2022 |
| POC-230 | Moorea TPC Relative Abundance | May 2022 |
| POC-246 | Moorea TPC Relative Abundance | May 2022 |
| POC-278 | Moorea TPC Relative Abundance | May 2022 |
| POC-279 | Moorea TPC Relative Abundance | May 2022 |
| POC-281 | Moorea TPC Relative Abundance | May 2022 |
| POC-285 | Moorea TPC Relative Abundance | May 2022 |
| POC-287 | Moorea TPC Relative Abundance | May 2022 |
| POC-289 | Moorea TPC Relative Abundance | May 2022 |
| POC-292 | Moorea TPC Relative Abundance | May 2022 |
| POC-294 | Moorea TPC Relative Abundance | May 2022 |
| POC-296 | Moorea TPC Relative Abundance | May 2022 |
| POC-257 | Moorea TPC Relative Abundance | May 2022 |
Bottom Row
| Sample ID | Project | Collection Date |
|---|---|---|
| POC-262 | Moorea TPC Relative Abundance | May 2022 |
| POC-263 | Moorea TPC Relative Abundance | May 2022 |
| POC-264 | Moorea TPC Relative Abundance | May 2022 |
| POC-265 | Moorea TPC Relative Abundance | May 2022 |
| POC-268 | Moorea TPC Relative Abundance | May 2022 |
| POC-306 | Moorea TPC Relative Abundance | May 2022 |
| POC-309 | Moorea TPC Relative Abundance | May 2022 |
| POC-311 | Moorea TPC Relative Abundance | May 2022 |
| POC-322 | Moorea TPC Relative Abundance | May 2022 |
| POC-329 | Moorea TPC Relative Abundance | May 2022 |
| POC-355 | Moorea TPC Relative Abundance | May 2022 |
| POC-356 | Moorea TPC Relative Abundance | May 2022 |
| POC-359 | Moorea TPC Relative Abundance | May 2022 |
| POC-360 | Moorea TPC Relative Abundance | May 2022 |
| POC-362 | Moorea TPC Relative Abundance | May 2022 |
| POC-363 | Moorea TPC Relative Abundance | May 2022 |
| POC-364 | Moorea TPC Relative Abundance | May 2022 |
| POC-365 | Moorea TPC Relative Abundance | May 2022 |
Gel 2
Top Row
| Sample ID | Project | Collection Date |
|---|---|---|
| POC-402 | Moorea TPC Relative Abundance | May 2022 |
| POC-406 | Moorea TPC Relative Abundance | May 2022 |
| POC Spawning 198 | positive control | POC Spawning 2022 |
| H20 | negative control | NA |
Bottom Row
| Sample ID | Project | Collection Date |
|---|---|---|
| POC-366 | Moorea TPC Relative Abundance | May 2022 |
| POC-367 | Moorea TPC Relative Abundance | May 2022 |
| POC-368 | Moorea TPC Relative Abundance | May 2022 |
| POC-369 | Moorea TPC Relative Abundance | May 2022 |
| POC-370 | Moorea TPC Relative Abundance | May 2022 |
| POC-372 | Moorea TPC Relative Abundance | May 2022 |
| POC-374 | Moorea TPC Relative Abundance | May 2022 |
| POC-379 | Moorea TPC Relative Abundance | May 2022 |
| POC-382 | Moorea TPC Relative Abundance | May 2022 |
| POC-383 | Moorea TPC Relative Abundance | May 2022 |
| POC-385 | Moorea TPC Relative Abundance | May 2022 |
| POC-386 | Moorea TPC Relative Abundance | May 2022 |
| POC-388 | Moorea TPC Relative Abundance | May 2022 |
| POC-392 | Moorea TPC Relative Abundance | May 2022 |
| POC-393 | Moorea TPC Relative Abundance | May 2022 |
| POC-399 | Moorea TPC Relative Abundance | May 2022 |
| POC-400 | Moorea TPC Relative Abundance | May 2022 |
| POC-401 | Moorea TPC Relative Abundance | May 2022 |
Gel Results
Bands of are present for the majority of samples but faint.
Will proceed with For primer sequencing for all samples with no dilution following sample cleanup.
Written on June 15, 2024