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Materials Required.

Working copy of the required plate or 4x4 glycerol grid
SD agar
Ampicillin aliquot
Tetracyclin aliquot
Hybaid colony plates or sterile bijou bottles for slopes
Disposable white plastic loops (for 96-well plates) or disposable yellow plastic needles (for 4x4 grids)

When the order arrives

1. Check that each order for clones is accompanied by the screening data sheet. If the screening data has been returned give the sheet to the HSO. If there is no data return sheet then go and check with the HSO, because it may have arrived earlier or been put on her desk. If no data has been supplied at all, then FAX the user and request that it be sent before the clone is sent out. There are FAX sheets already typed up for this. The clone can be issued when the data is sent in.

NB; we do not normally require screening data for the CEPH MegaYAC library, as the information is usually obtained from literature. If in doubt check with the HSO.

2. Sometimes the clone requests have to be 'translated' as the co-ordinates of the rows and columns have been given. Write in the clone ID.
3. Any requests for mouse YAC clones, please refer to the SO.

Pouring the agar slopes or plates

Usually there should be stocks of plates or slopes in the 4^oC cabinet. A good time to make slopes is when there is free time during a day. Slopes will keep for about 3 weeks, but plates only for 2 weeks maximum.
All YAC clones are streaked out onto SD agar.

1. Melt the SD agar. One 500ml bottle will be enough to pour out 100 slopes (+ some extra plates) or 12-14 Hybaid colony picking plates.
   Loosen the top of the SD bottle and place in the microwave oven (Toshiba, ER-8730)
   DO NOT PUT METAL LIDS IN THE MICROWAVE.
   The microwave power should be set at 3 for one bottle, and at 5 for more than one bottle. Initially set the timer for twenty minutes, and after that for however many more minutes you think will be needed to melt the agar. Check whether the agar is fully melted by tightening the lid and gently turning the bottle upside down whilst holding it up to the light.

ALWAYS HANDLE THE HOT AGAR BOTTLES USING THE RED RUBBER 'HOT HAND', OR APPROPRIATE LEATHER GLOVES.

2. When melted, place the agar bottle in the Hybaid hybridisation oven, with the temperature set to 65^oC. This is to allow the agar to cool, but not low enough to set, so that the antibiotics are not destroyed by too high a temperature when they are added.
3. Thaw one 500µl aliquot of ampicillin and one 1.25ml aliquot of tetracycline for each 500ml bottle of SD agar.
4. Switch on the YAC laminar flow cabinet (the 'hood') and spray all the surfaces with 70% alcohol.
5. Take the SD agar to the cabinet and add the antibiotics, using a Gilson pipette and autoclaved tips. All used tips and small tubes should be disposed of in a sweetie jar.

Slopes

Take the bijou bottles to the hood, and take off the lids of the bottles, placing them on the cleaned hood surface.
Place the bottles in one of the clear plastic 96-well moulds for agar slopes.
Take a row of 10 bottles and place them in the LHS of another plastic mould, with all the labels facing the RHS.
Pour some of the agar into a sterile 50ml Falcon tube, and use this as a 'beaker' to pour into the bijou bottles. The exact volume does not matter, but aim to fill them slightly more than half full. When the first row is filled, place bijou's in the second row and fill as before.
When 5 rows are filled, carefully tilt the whole rack up and lean it against the side of the hood. Prop up the base with a 'hedgehog' so that it doesn't slide down. Aim for the greatest slope that you can, without the agar dropping out, and with the slope covering the label (i.e.; the labels that were all pointed towards the RHS when pouring are now pointing down to the base of the hood.
Now start on the second 5 rows in exactly the same way, and at the end, tilt and prop this mould up also.
Leave to set.
There will be some agar left over and this can be used to pour out a few Hybaid colony picking plates.
When the slopes are set, replace the lids, still working within the hood, and store, with the date on, in the 4^oC cabinet.

Plates

Open a pack of sterile Hybaid colony picking plates in the hood, and place them at the back and the sides of the hood. Put the base of the plate on the top of its face-down lid. (the base of our hood is not flat, so if we place plates in the centre we get very unlevel agar)
Pour the agar into the plates. The volume is not too critical, but it is usual to fill 12-14 plates from each bottle of agar.
If there are any air bubbles on the plates, then take a sterile loop or needle and move the bubble to the side of the plate before the agar has started to set.
When the plates are set, replace the lids, and wrap the stack of plates in clingfilm and date, before placing in the 4oC cabinet.
After the plates are set they will start to dry in the hood when the lids are off. It is useful to let the surface of the agar start to dry to reduce subsequent condensation, but they should not be allowed to dry out to much which will lead to cracking of the agar.

Streaking the clones using the working copy of the library

Make a list of all the clones requested in your lab. daybook.
If a user has requested 8 or more clones, these can be streaked out onto a plate which can take 16 clones. There are printed sheets with the 16 divisions already marked off. Write in the names of the clones and then stick this sheet to the bottom of the agar plate.
For 8 or less clones use agar slopes, and write the clone name on the label.
Use your list of clones to take out the corresponding working copies of the plates out of the -70^oC freezer. Leave the plates to thaw on the bench.
The clones can be streaked when the plates are thawed.
Mop the plate before taking to the hood.
Use a sterile white loop (only open the packet in the hood) and go into the well containing your required clone. put the loop right to the bottom of the well because the cells will have settled there.
Take the loop out and touch the surface of the agar, with the flat side of the loop against the agar. Stroke the surface of the agar from side to side. If you are streaking onto a plate, make sure that you stay within the divisions that mark out the clone's position on the plate
Replace the lid on the working plate copy, before the plate is removed from the hood.

When all the clones have been streaked, the slopes and the plates should be put in the 30^oC incubator. The plates should be wrapped in clingfilm and placed upside down, so that any condensation from the lid does not drop onto the clones and smear them.

The working copy plates should be refrozen at -70^oC.

Streaking the clones using the 4x4 glycerol plates.

Make a list of all the clones requested in your lab. daybook.
If a user has requested 8 or more clones, these can be streaked out onto a plate which can take 16 clones. There are printed sheets with the 16 divisions already marked off. Write in the names of the clones and then stick this sheet to the bottom of the agar plate.
For 8 or less clones use agar slopes, and write the clone name on the label.

The 4x4 glycerol plates are stored at -20^oC.

There are 16 source plates on each 4x4 glycerol plate, so often you will need a glycerol plate for more than one clone.
Locate your required clone by using the record sheets that are stored in each YAC library folder.

Here is an example:

Clone required = 623 G8

Look at the top square to see exactly where plate 623 is located on the 4x4 grid
It is on the third row down and is the third position across
Now look at your 4x4 glycerol and identify position G8 - you will see that each group of 16 small clones occupies the same position as on a 96-well plate
Go to that position, and using a sterile yellow needle, carefully pick off the small clone that is in the third row down and is in the third position across.
Streak the clone out onto agar, exactly as you would do with a loop from a liquid well. The needle will not move quite so easily across the agar, but with a series of gentle strokes you will transfer the clone to the agar.
Incubate at 30^oC exactly as before.
Cross off the position 623 G8 on the record sheet to indicate that there is no longer a clone at that position.
Place the 4x4 plate back in the -20oC freezer and remove the next 4x4 plate if required.

Incubating the clones

YAC clones are usually grown for 2 days at 30^oC.
There are exceptions to this:

After the two days growth, the clones are removed from the incubator and checked for growth. If they are all growing well, then they are matched up with the correct user request form.
All slopes for one user are placed in a plastic bag, and the bag labelled with the user request number, so that staff in the post room can match up the orders with the correct user to whom they will be posted.
All plates are wrapped at the edge with parafilm, and then wrapped in bubble wrap and sellotape, to protect them in the post and prevent the lid from coming off. Again, the request number is written on the edge of the package.
Each completed request form should have a new form attached.
All orders - request forms and streaked clones should be taken to the office for postage later in the day. The earlier it can be delivered to the office, the easier the task for the office staff.

If a clone is found not to grow - check the record sheets for that library. It may well be that well position is actually empty. In which case inform the user, on their request form, that we do not have this clone. This is often the case with the CEPH mega-YAC library, and we have a printed sheet that can be sent to the user, describing the loss of clones during the mega-YAC clean-up.

If the clone should have grown, but didn't, restreak the clone. If this fails consult the HSO.

Every effort should be made to despatch clone requests within 2-4 days of receipt (other work permitting)

Guidelines prepared for CABRI by HGMP, December 1998
Page layout by CERDIC
Copyright CABRI, 1998