June 3rd, 2021
RPA Workflow
Done:
- Preliminary temperature optimization (A2)
- 37ºC...2F, 10R
- 42ºC...4F, 11R
To-Do:
- Repeat temperature optimization (A2)
- Optimize timing (20- 40 min)
- Repeat primer + temperature + timing optimization (A2)
- Try a real sample
- Try with the heat block
The TwistDX RPA protocols will be used for reaction experiment.
Today: Made 10mL of Chloramphenicol stock aliquoted into 1000µL portions.
Via updated “Antibiotic Stock Protocol”
In Wiki: Update “Antibiotic Stock Protocol – (1000 x 1 µL/mL Media) – 10mL Solutions”
Cm 1. Dissolve 0.25g of Chloramphenicol (25mg/mL) in reagent ethanol 10mL
- Store aliquots 1000µL into microcentrifuge tubes for storage at -20ºC
To Do:
- Repeat experiment in Wiki RPA Figure 6 – Both primer pairs at both temperatures (40 min)
- PCR Cleanup
- Gel for product verification
June 7th, 2021
RPA Figure 6 Re-do
The reaction will be set up using the protocol posted on the iGEM Teams Files.
| Reagent | 1 Reaction | 10 Reactions |
|---|---|---|
| 2x Reaction Buffer | 25µL | 250µL |
| 10mM dNTPs | 1µL | 10µL |
| Nuclease-free Water | 8.2µL | 82µL |
| 10x Probe E-mix | 5µL | 50µL |
| --- (Add 20x Rxn mix to lid of Mastermix, invert 10 times and spin) --- | ||
| 20x Core Reaction Mix | 2.5µL | 25µL |
- 2.5 µL of MgOAc to be added to each reaction.
- 1.0 µL of (DNA, water, or control DNA) to be added to each reaction.
PCR Cleanup:
- 50µL Product
- 30µL elution buffer
Gel: 0.4% agarose 50mL TBE 1x
- 1kb marker
- 2F,10R samples: 5µL eluted product, 1µL of 6x Loading Dye
- 4F,11R samples: DILUTED: 2µL eluted product, 8µL Nuclease-free water, 2µL of 6x Loading Dye