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
June 8th, 2021
Repeat Experiment from June 6th
The following samples had their nanogram concentrations recorded in the table below.
*Gel 10µL of unclean RPA product 1kb + MWL measured up at where the product should be
June 9th, 2021
RPA Reaction Cleaned up DNA Core
Nanogram concentrations of samples were recorded
June 10th, 2021
RPA Clean Trial 3
Nanogram concentrations of samples were recorded
June 16th, 2021
Repeat Experiment from June 7th using a NEW Twist-DX Kit
- 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.
- Samples were incubated for 40 minutes at their respective temperatures.
PCR Cleanup:
- 45µL RPA Product
- 40µL elution buffer
Gel: 0.4g agarose 50mL 1x TBE
- 1kb ladder
- 2F,10R samples: DILUTED: 2µL eluted product, 8µL Nuclease-free water, 2µL of 6x Loading Dye
- 4F,11R samples: DILUTED: 2µL eluted product, 8µL Nuclease-free water, 2µL of 6x Loading Dye
- 10µL of each sample mixture to each well
To Do:
- Repeat gel using clean RPA product, do NOT dilute.
June 23rd, 2021
Repeat Experiment from June 7th using a NEW Twist-DX Kit
New dNTP Stock Solution to be prepared...
| Reagent | Stock | Working |
|---|---|---|
| dNTPs | 100mM | 10mM |
$$(100mM)(v_{1}) = (10mM)(100µL)$$ $$v_{1} = 10µL$$
10µL of each dNTP stock will be added, in addition to 60µL of nuclease-free water to bring the final volume to 100µL
New working primers will be prepared.
diluted primers
- 10_RPA_R
- 11_RPA_R
- 4_RPA_F
- 2_RPA_F
100mM -> all diluted with 10µL of primer + 90µL of nuclease-free (NF) water
The following set up will be used: