This project is part of an attempt to repeat SHERLOCK protocol Kellner at al (2019) Nature protocols in TSL. It started as a side project carried out during Corona virus lockdown.
- RNAseAlet kit test
- Test with of and synthetic RNA
- Use Isothermal amplification (SHERLOCK)
- Test with a real sample
All the data are shared on GitHub here
Link to experimental details - pipetting spreadsheet
### Direct links to the results
Spectrofluorimeter, 96-well format
Test 01: Test of the old kit
The old kit is dead!
Test 02: Fresh substrate works
New substrate works!
Test 03: DEPC inhibitor works
DEPC inhibition works!
Test 04: Cas13 with guide and target RNA
This did not work, repeat it with minor changes.
This did not work either.
Test 06-2 benzoase: New batch of Cas13 with guide and target DNA - benzoase treated and remeasured
Something is wrong with the detection assay. We need to test the substrate and buffer, possible other components.
Test 07 substrate: Test of different substrate batches in RNAaseAlert assay
Clearly we have to use more of substrate than is stated in Kellner's protocol. is our spectrofluorimeter working well?
Test 07-2 buffer: Test of different buffers in RNAaseAlert assay only
Yes, buffer has influence on the assay. Again, is spectrofluorimeter all right?
Test 08-1: Test of DNA target
Test 08-2: Test of DNA target 24h later
Something must be fundamentally wrong.
Yes, the protein is the problem.
Sequencing of the original plasmid revealed, that an incorrect protein was expressed. When we rectify the problem, the next test should be decisive.
Test 11-2: New "correct" Cas13a
Partial success!!
Test 12: Fluorescein test
We have a sensitivity issue with our plate reader or general set up such as plate type, volume, settings.
Test 13: Orf1ab dilutions in 384-well plate
It starts to look good, despite the sensitivity is still not what they publish.
Test 18: Josie #4, 200819
Trying to find quantitative variable - gradient vs. sum of all data points.