Touchdown PCR for ITS2
Touchdown PCR for ITS2 processing
The goal of touchdown PCR (Korbie and Mattick 2008)is to increase specificity of the primers during PCR. I am currently having troubles with our original ITS2 protocol on coral samples with very low Symbiodiniaceae densities. Our previous protocol is sequencing coral DNA when the Symbiodiniaceae densities are very low.
Previous gel results:
G3 (Vial 43) is higher than the other samples.
Samples 74, 41, 40 had very faint bands.
20220315
This protocol I used from Korbie and Mattick 2008 and used a Tm of 52°C. In short, this protocol starts 10°C+Tm and decreases 1°C every cycle until it reaches Tm.
Samples
I am using DNA that is already diluted to 4ng/uL in this post
| Fragment_ID | Group | Vial | Well_dilution_plate |
|---|---|---|---|
| R8-52 | Bleached | 43 | G3 |
| R37-52 | Bleached | 74 | A6 |
| R35-52 | Partial-Mortality | 41 | F3 |
| R36-52 | Partial-Mortality | 40 | E3 |
| R7-52 | Control | 34 | H2 |
Master Mix Calculations
Determining number of wells:
| 20220315 test set | |
|---|---|
| # Samples | 4 |
| Positive Control | 1 |
| Negative Control | 1 |
| Error | 1 |
| Total # Samples | 7 |
| Total # Wells | 14 |
Calculating Master mix:
| Volume (uL) | Total # Wells | Master Mix Vol (uL) | |
|---|---|---|---|
| Phusion Master Mix | 12.5 | 14 | 175 |
| F Primer (10µM) | 0.5 | 14 | 7 |
| R Primer (10µM) | 0.5 | 14 | 7 |
| Ultra Pure H2O | 10.5 | 14 | 147 |
| Total Vol of MM | 336 | ||
| 24 uL/Well | 14 |
PCR settings
| Phase | Step Number | Number of cycles | Step | Temperature | Time |
|---|---|---|---|---|---|
| 1 | 1 | 1 | Denature | 95 | 3 min |
| 1 | 2 | 1 | Denature | 95 | 30 s |
| 1 | 3 | 1 | Anneal | 62 | 45 s |
| 1 | 4 | 1 | Elongate | 72 | 60 s |
| 1 | 2 | 1 | Denature | 95 | 30 s |
| 1 | 3 | 1 | Anneal | 61 | 45 s |
| 1 | 4 | 1 | Elongate | 72 | 60 s |
| 1 | 2 | 1 | Denature | 95 | 30 s |
| 1 | 3 | 1 | Anneal | 60 | 45 s |
| 1 | 4 | 1 | Elongate | 72 | 60 s |
| 1 | 2 | 1 | Denature | 95 | 30 s |
| 1 | 3 | 1 | Anneal | 59 | 45 s |
| 1 | 4 | 1 | Elongate | 72 | 60 s |
| 1 | 2 | 1 | Denature | 95 | 30 s |
| 1 | 3 | 1 | Anneal | 58 | 45 s |
| 1 | 4 | 1 | Elongate | 72 | 60 s |
| 1 | 2 | 1 | Denature | 95 | 30 s |
| 1 | 3 | 1 | Anneal | 57 | 45 s |
| 1 | 4 | 1 | Elongate | 72 | 60 s |
| 1 | 2 | 1 | Denature | 95 | 30 s |
| 1 | 3 | 1 | Anneal | 56 | 45 s |
| 1 | 4 | 1 | Elongate | 72 | 60 s |
| 1 | 2 | 1 | Denature | 95 | 30 s |
| 1 | 3 | 1 | Anneal | 55 | 45 s |
| 1 | 4 | 1 | Elongate | 72 | 60 s |
| 1 | 2 | 1 | Denature | 95 | 30 s |
| 1 | 3 | 1 | Anneal | 54 | 45 s |
| 1 | 4 | 1 | Elongate | 72 | 60 s |
| 1 | 2 | 1 | Denature | 95 | 30 s |
| 1 | 3 | 1 | Anneal | 53 | 45 s |
| 1 | 4 | 1 | Elongate | 72 | 60 s |
| 1 | 2 | 1 | Denature | 95 | 30 s |
| 1 | 3 | 1 | Anneal | 52 | 45 s |
| 1 | 4 | 1 | Elongate | 72 | 60 s |
| 2 | 1 | 25 | Denature | 95 | 30 s |
| 2 | 2 | 25 | Anneal | 52 | 45 s |
| 2 | 3 | 25 | Elongate | 72 | 60 s |
| 3 | 1 | 1 | Elongate | 72 | 5 min |
| 3 | 2 | 1 | Halt | 4 | Hold |
Gel Results
Today I ran a 1% gel on a 100 well gel.
This protocol did not seem to work based off of the gel results.
20220317
Today I ran 2 different variations of the thermocycler settings:
-
TD_2: Starting 10°C + Tm, going down a degree every cycle until Tm-5°C and continuing the remaining cycles at Tm-5°C.
-
TD_3: A protocol modified from H. Putnam, 7 min at 95°C, 20 cycles of 94°C for 30s, 62°C for 30s (decreased by 0.5°C at each cycle), 72°C for 1 min, followed by 12 additional cycles with an annealing temperature set at 52°C, final extension of 72°C for 7 min.
TD_2 thermocycler settings
| Phase | Step Number | Number of cycles | Step | Temperature | Time |
|---|---|---|---|---|---|
| 1 | 1 | 1 | Denature | 95 | 3 min |
| 1 | 2 | 1 | Denature | 95 | 30 s |
| 1 | 3 | 1 | Anneal | 62 | 45 s |
| 1 | 4 | 1 | Elongate | 72 | 60 s |
| 1 | 2 | 1 | Denature | 95 | 30 s |
| 1 | 3 | 1 | Anneal | 61 | 45 s |
| 1 | 4 | 1 | Elongate | 72 | 60 s |
| 1 | 2 | 1 | Denature | 95 | 30 s |
| 1 | 3 | 1 | Anneal | 60 | 45 s |
| 1 | 4 | 1 | Elongate | 72 | 60 s |
| 1 | 2 | 1 | Denature | 95 | 30 s |
| 1 | 3 | 1 | Anneal | 59 | 45 s |
| 1 | 4 | 1 | Elongate | 72 | 60 s |
| 1 | 2 | 1 | Denature | 95 | 30 s |
| 1 | 3 | 1 | Anneal | 58 | 45 s |
| 1 | 4 | 1 | Elongate | 72 | 60 s |
| 1 | 2 | 1 | Denature | 95 | 30 s |
| 1 | 3 | 1 | Anneal | 57 | 45 s |
| 1 | 4 | 1 | Elongate | 72 | 60 s |
| 1 | 2 | 1 | Denature | 95 | 30 s |
| 1 | 3 | 1 | Anneal | 56 | 45 s |
| 1 | 4 | 1 | Elongate | 72 | 60 s |
| 1 | 2 | 1 | Denature | 95 | 30 s |
| 1 | 3 | 1 | Anneal | 55 | 45 s |
| 1 | 4 | 1 | Elongate | 72 | 60 s |
| 1 | 2 | 1 | Denature | 95 | 30 s |
| 1 | 3 | 1 | Anneal | 54 | 45 s |
| 1 | 4 | 1 | Elongate | 72 | 60 s |
| 1 | 2 | 1 | Denature | 95 | 30 s |
| 1 | 3 | 1 | Anneal | 53 | 45 s |
| 1 | 4 | 1 | Elongate | 72 | 60 s |
| 1 | 2 | 1 | Denature | 95 | 30 s |
| 1 | 3 | 1 | Anneal | 52 | 45 s |
| 1 | 4 | 1 | Elongate | 72 | 60 s |
| 1 | 2 | 1 | Denature | 95 | 30 s |
| 1 | 3 | 1 | Anneal | 51 | 45 s |
| 1 | 4 | 1 | Elongate | 72 | 60 s |
| 1 | 2 | 1 | Denature | 95 | 30 s |
| 1 | 3 | 1 | Anneal | 50 | 45 s |
| 1 | 4 | 1 | Elongate | 72 | 60 s |
| 1 | 2 | 1 | Denature | 95 | 30 s |
| 1 | 3 | 1 | Anneal | 49 | 45 s |
| 1 | 4 | 1 | Elongate | 72 | 60 s |
| 1 | 2 | 1 | Denature | 95 | 30 s |
| 1 | 3 | 1 | Anneal | 48 | 45 s |
| 1 | 4 | 1 | Elongate | 72 | 60 s |
| 1 | 2 | 1 | Denature | 95 | 30 s |
| 1 | 3 | 1 | Anneal | 47 | 45 s |
| 1 | 4 | 1 | Elongate | 72 | 60 s |
| 2 | 1 | 20 | Denature | 95 | 30 s |
| 2 | 2 | 20 | Anneal | 47 | 45 s |
| 2 | 3 | 20 | Elongate | 72 | 60 s |
| 3 | 1 | 1 | Elongate | 72 | 5 min |
| 3 | 2 | 1 | Halt | 4 | Hold |
TD_3 thermocycler settings
| Phase | Step Number | Number of cycles | Step | Temperature | Time |
|---|---|---|---|---|---|
| 1 | 1 | 1 | Denature | 95 | 7 min |
| 1 | 2 | 1 | Denature | 94 | 30 s |
| 1 | 3 | 1 | Anneal | 62 | 30 s |
| 1 | 4 | 1 | Elongate | 72 | 60 s |
| 1 | 2 | 1 | Denature | 94 | 30 s |
| 1 | 3 | 1 | Anneal | 61.5 | 30 s |
| 1 | 4 | 1 | Elongate | 72 | 60 s |
| 1 | 2 | 1 | Denature | 94 | 30 s |
| 1 | 3 | 1 | Anneal | 60 | 30 s |
| 1 | 4 | 1 | Elongate | 72 | 60 s |
| 1 | 2 | 1 | Denature | 94 | 30 s |
| 1 | 3 | 1 | Anneal | 59.5 | 30 s |
| 1 | 4 | 1 | Elongate | 72 | 60 s |
| 1 | 2 | 1 | Denature | 94 | 30 s |
| 1 | 3 | 1 | Anneal | 59 | 30 s |
| 1 | 4 | 1 | Elongate | 72 | 60 s |
| 1 | 2 | 1 | Denature | 94 | 30 s |
| 1 | 3 | 1 | Anneal | 58.5 | 30 s |
| 1 | 4 | 1 | Elongate | 72 | 60 s |
| 1 | 2 | 1 | Denature | 94 | 30 s |
| 1 | 3 | 1 | Anneal | 58 | 30 s |
| 1 | 4 | 1 | Elongate | 72 | 60 s |
| 1 | 2 | 1 | Denature | 94 | 30 s |
| 1 | 3 | 1 | Anneal | 57.5 | 30 s |
| 1 | 4 | 1 | Elongate | 72 | 60 s |
| 1 | 2 | 1 | Denature | 94 | 30 s |
| 1 | 3 | 1 | Anneal | 57 | 30 s |
| 1 | 4 | 1 | Elongate | 72 | 60 s |
| 1 | 2 | 1 | Denature | 94 | 30 s |
| 1 | 3 | 1 | Anneal | 56.5 | 30 s |
| 1 | 4 | 1 | Elongate | 72 | 60 s |
| 1 | 2 | 1 | Denature | 94 | 30 s |
| 1 | 3 | 1 | Anneal | 56 | 30 s |
| 1 | 4 | 1 | Elongate | 72 | 60 s |
| 1 | 2 | 1 | Denature | 94 | 30 s |
| 1 | 3 | 1 | Anneal | 55.5 | 30 s |
| 1 | 4 | 1 | Elongate | 72 | 60 s |
| 1 | 2 | 1 | Denature | 94 | 30 s |
| 1 | 3 | 1 | Anneal | 55 | 30 s |
| 1 | 4 | 1 | Elongate | 72 | 60 s |
| 1 | 2 | 1 | Denature | 94 | 30 s |
| 1 | 3 | 1 | Anneal | 54.5 | 30 s |
| 1 | 4 | 1 | Elongate | 72 | 60 s |
| 1 | 2 | 1 | Denature | 94 | 30 s |
| 1 | 3 | 1 | Anneal | 54 | 30 s |
| 1 | 4 | 1 | Elongate | 72 | 60 s |
| 1 | 2 | 1 | Denature | 94 | 30 s |
| 1 | 3 | 1 | Anneal | 53.5 | 30 s |
| 1 | 4 | 1 | Elongate | 72 | 60 s |
| 1 | 2 | 1 | Denature | 94 | 30 s |
| 1 | 3 | 1 | Anneal | 53 | 30 s |
| 1 | 4 | 1 | Elongate | 72 | 60 s |
| 1 | 2 | 1 | Denature | 94 | 30 s |
| 1 | 3 | 1 | Anneal | 52.5 | 30 s |
| 1 | 4 | 1 | Elongate | 72 | 60 s |
| 1 | 2 | 1 | Denature | 94 | 30 s |
| 1 | 3 | 1 | Anneal | 52 | 30 s |
| 1 | 4 | 1 | Elongate | 72 | 60 s |
| 1 | 2 | 12 | Denature | 94 | 30 s |
| 1 | 3 | 12 | Anneal | 52 | 30 s |
| 1 | 4 | 12 | Elongate | 72 | 60 s |
| 3 | 1 | 1 | Elongate | 72 | 7 min |
| 3 | 2 | 1 | Halt | 4 | Hold |
Samples Master Mix calculations
I used the same samples and master mix calculations for each thermocycler setting.
Gel Results
