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Latest revision as of 08:52, 10 September 2015
DNA + Cellfree Expression on PDMS
protocol for spotting of DNA and cellfree expression on PDMS flow cells
protocol according to AG Roth
material: chemicals, used kits, …
duration: … min
Spotting DNA
- Spot DNA (c= 25 ng/µl) on flow cell (spot size can be 0.5 µl or more)
- Incubate over night at room temperature in humid atmosphere in petri dish sealed with Parafilm
- Heat whole petri dish in oven at 60°C for 30min in order to dry the DNA (leave humid tissue inside and make sure the petri dish is still sealed)
- put the flow cell in a petri dish filled with H2O and wash it
DNA has bound to surface if water drops appear on it during washing step
- dry flow cell with wafergun
Storage of spotted flow cells
- store in fridge sealed with parafilm without humid tissue
Cellfree Expression "on air"
- Put flow cell with its frontside (with spots) on a clean glass slide
- Make sure the flow cell does not collapse!
- Add a second glass slide orthogonally to the first one on the backside of the flow cell to give some stability (should look like a cross now)
- Prepare 15 µl cellfree Expression mix per flow cell
AG Roth Mix | ||
---|---|---|
component | concentration | volume |
H2O | 3,75 µl | |
Buffer | 6 µl | |
Lysate | 5,25 µl |
- Put flow cells in petri dish with humid tissue and sealed with parafilm
- Incubate at 37°C
- for 25 min (for a quick check)
- for 2 h (for full Expression)
- Measure fluorescence in microscope of AG Eimer (only possible with Tobi)