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LABORATORY PROCEDURES FOR GENOMIC LIBRARIES

REFERENCE NO: GLI/1998/3/12

The agar plates can be poured in advance and stored in the 4^oC cold cabinet. (see GLI/1998/3/2 for method)

1. Load the tool onto the tool holder. The end with the locking bolt goes towards the back, with the clip at the back pulled out to the left. When the tool is fully loaded, and is flush with the front of the holder, slide across the clip at the back to engage the bolt, so that the tool is locked in. Close the door.
2. Double click the mouse on the gridder icon and the robot will switch on the correct main programme. The programme begins with the tool going to its home position at the back LHS and then back to the loading position. Make sure the laminar flow air supply box on top of the hood is switched on.
3. Click on the file menu at the top of the screen and drag down till the "open" instruction is highlighted. When the saved files are displayed highlight the programme called 4x41glyc.grd and then the programme that we want will be loaded. This programme uses 16 source plates and one destination plate, in a 4x4 grid pattern with the 96 fine pin head.
   The sterilisation steps are:

bath 3 (70% alcohol)	10 secs
bath 4 (heater)	5 secs
air dry	15 secs

NB. A quicker way to load the programme, if you have used it recently, is to highlight it when it is shown in the file menu, among the list of the four most recently used programmes.

4. When you are satisfied that the programme looks correct try a dummy run by clicking on run programme. The programme should ask you if you want to start at spot 1, plate 1. If it does, click OK, if not (because a previous run was interrupted) reset the two figures to 1. Whilst the robot is doing its dummy run check that it is doing everything, as expected, correctly.
5. At the end of the test run, open the robot and fit the Hybaid plate for the alcohol bath. This is in the position in front of the heater. Make sure the plate is really securely in position before adding the 70% alcohol, which should 3/4 fill the bath. spray the black plastic base on the LHS with 70% alcohol.
6. Load the first 8 source plates into the rack. It is very important that the plates are clipped fully into the rack. When the rack is placed on the LHS position in the robot, check all plates are correctly loaded, by looking at them at eye level. An incorrectly placed plate will be higher than the correctly placed ones.
7. Place the destination plate, with the Hybond membrane into the first position on the second rack. These colony picking plates are larger than the 96-well plates and are much stiffer fitting into the racks. But it is essential that they are fully seated in the rack, otherwise they may move during gridding and the gridded filter pattern will be very haphazard.
8. Carefully remove the lids from the trays, placing them on the black plastic base on the LHS in the robot. Remove the lids starting with the plates furthest away, to avoid putting your arms over the open plates (and replace the lids, after gridding, in the opposite order). Remove the lid from the agar destination plate. Shut the door.

Robot arrangement:

	Source Plates			Gridded Plates
	1	5		X
	2	6
	3	7
	4	8						Heater
								EtOH

9. Start the programme.
   After the first 8 plates have been completed, the programme will ask you to load the next source plates (9-16). Remove the plates 1-8 after replacing the lids, load plates 9-16, remove the lids, shut the robot door, and press the OK icon. The robot will now complete gridding the second half of the gridded filter.
10. Sometimes the programme will stop halfway through. Click the OK button, and the gridder will return to its start-up screen. Re-open the gridder programme. If the programme says 'speed out of range', press the abort icon and your original programme will be recovered. When you restart the programme you will be asked if you want to start at spot 'x'. Usually the robot will have already gridded from this spot, and if you have observed this it is safe to enter the next position and proceed from there. If you are not sure, then proceed from the suggested spot and the worst that will happen is that there will be a double spot (of the same clone) on that position. Either way the tool will be sterilised before it goes to the next plate.
11. Repeat this cycle, 7-9, for however many gridded membranes you are preparing.
12. At the end of the session:

-refreeze the source plates
-incubate the gridded membrane plates
-time permitting, re-sonicate the tool ready for the next person.


Incubating the gridded membranes.

Place the agar plates, upside down, wrapped in cling film, in the 30^oC incubator for two days.


Placing the membranes on paper soaked in glycerol.

1. Add an equal volume of SD or YPD broth to a Duran bottle of autoclaved glycerol. Add ampicillin and tetracycline to the required final concentration. E.g.;
   100ml glycerol
   add 100ml broth
   + 200µl ampicillin
   + 500µl tetracycline
2. Work in the YAC 'hood'. Pour some of the 50% glycerol into a sterile large petri dish. Sterilise the blue-handled forceps by flaming in alcohol, and then use them to remove a sheet of pre-cut, autoclaved 3MM paper and soak the paper in the glycerol. Lift out the paper with the forceps and allow the glycerol to drain off. Just before it has stopped dripping lay the paper into a Hybaid colony picking plate, taking care to ensure that there are no air bubbles underneath the paper. If there are air bubbles, the sheet can be lifted off and relaid.
   The glycerol amount can be adjusted if it is incorrect, by either draining off excess glycerol by tipping the plate, or by adding extra glycerol if the paper is too dry.
3. Label this plate, on the lid, with:

the library name
the date
plate numbers contained on the grid

Also place a red line around the lid and the base in the LH corner.

4. Hold the agar plate with the gridded membrane, and carefully lift the membrane off using the forceps. This is best achieved by using forceps to lift up the RHS of the membrane, lifting the membrane off the agar and dragging it across the plate until you can grasp the LHS of the membrane with the second pair of forceps. Carefully place the membrane onto the glycerol soaked paper, laying it in such a way as to prevent air bubbles underneath. When using the forceps to lift the membrane you must be very careful not to touch any of the colonies. If there are any air bubbles trapped, try lifting the membrane and relaying it.
5. The plates are now ready to be frozen, RIGHT SIDE UP, in the -20^oC freezer. Throw away the old 4x4 plates that you are replacing.
6. The final job is to complete new record sheets and to place them in the appropriate folder.

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Site maintained by Paolo Romano. Last revised on February 2023.