Template:SBB- Streptavidin SafeImager Assay

From OpenWetWare
Jump to: navigation, search

Positive control is pBca9145-Bca9494 {AraC-Pbad}{rbs.cpx} (this also contains a strep tag and has been tested for display)

Negative control is pBca9495CA-Bca1363 {Pbad.rbs.prepro.StrepTag}


1. Grow cells to saturation and induce with arabinose. Typically, inoculate from an existing culture 1:1000 or from a colony and incubate overnight. In the morning, use last night's saturated culture to inoculate 1:100 cell dilution. Add arabinose (2.0 mg/mL or 100 μg/mL final concentration has been shown to work) to the new culture and assay in the afternoon (~ 5 hours). Before assaying, measure OD600.

2. Add 25 uL of cells, 75 uL of PBS, and 1.0 uL of Streptavidin-PE (0.5 mg/mL) to wells in a V-bottomed 96-well plate. This assay seems to be sensitive either to the number of cells (?) or the media/PBS ratio (pH?), so don't stray far from 1:3.

3. Seal well plate with a film and incubate at 37C without shaking for 30 minutes.

4. Pellet cells by spinning down plate at 5000 RPM for 5 minutes.

5. Invert plate very gently and visualize cells on the SafeImager using the filter plate. Photograph the plate (TIF or RAW preferably).

6. Open the image in ImageJ. Go to Image > Type > 8-bit.

7. Go to Analyze > Set Measurements and make sure that "Integrated Density". is checked.

8. Use the Ellipse tool to draw a circle around one of your pellets. If your circle is too large it will pick up a lot of noise from the plastic well. Click Control-M to measure the integrated density of your selection - a spreadsheet will open containing your data.

9. Drag the ellipse to the next pellet. (Do not redraw the ellipse - that will give you messy data.) Click Control-M. Repeat until you've measured every pellet.