Four Corners Optimization
The traditional way of preparing optimization screens is making highly concentrated stock solutions of all the ingredients and the dispensing them individually to obtain the desired gradients throughout the plate.
An alternative is four-corners optimization. Starting with stock solutions corresponding to the four corner solutions of the desired grid allows for easier automization because it avoids high viscosity. This is now implemented on the Tecan.
The four corner solutions are defined as this: A – Top left corner, B – top right corner, C – bottom left corner, D – bottom right corner.
Four-corners optimization is possible with a large number of trays. You can pick from:
- 24-well plates – This can be sitting-drop or hanging-drop plates. Note that there are four columns and six rows (rotated by 90 degrees from how most people consider these plates).
- 48-well MRC Maxi plates – These have the footprint of 96-well plates but can accommodate drops up to 5 µl big. Note that there are six columns and eight rows.
- 48-well MRC Maxi plates with two grids – This sets up two different 24-well optimization grids in one 48-well plate. You have to pick up eight troughs and provide two sets of corner solutions.
- 96-well MRC plates – These can be used with the Mosquito and correspond to the picture above. Note that accurately pipetting 1.1 µl (well B11 and F11) with the Tecan is a bit of a stretch.
- 96-well MRC plates in quadruplicate – This layout puts a 24-well grid in quadruplicate. Not sure if this makes sense, but it trades the extremely fine gradation of the regular 96-well tray for reproducibility. If you get crystals, chances are you have four drops with them.
- 96-well triple-drop plates – For those wanting to try their protein in more concentrations than is possible with MRC plates, we have triple-drop plates. They can be used with the Mosquito.
You can also set up one-dimensional grids, possibly two different ones covering half a tray each, or pretty much anything you can come up with. The Tecan is very flexible.
Please book these optimization screens on Sharepoint. I have added two fields towards the end of the list, for 96-well screens (charged an empty plate + charge-out) and 48-well screens (anything else where you provide the plate, chargeout only).
- You have to provide the 48-well plates. I don't have any.
- You have to provide 10 ml of each of the corner solutions.
- pH gradients won't necessarily be linear.
- The 96-well tray is made with a tiny volume of one of the corner solutions in some wells. The gradient might thus not be perfect. The 48-well grids are all right.
- You have to come up here before the experiment and borrow 4 Tecan troughs for each grid you want to set up. The troughs are reused, but I can't wash them up here, so you'll have to do that.
- The facility has a plate shaker for mixing after the tray has been set up.