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LABORATORY PROCEDURES FOR GENOMIC LIBRARIES

REFERENCE NO: GLI/1998/3/2


TITLE: PRODUCTION OF YAC CELL POOLS FOR SPHEROPLASTING



Materials

Sterile round petri dishes [Falcon 1058] if 'stamping' by hand or
Hybaid colony picking plates [Hybaid HB CPP] if 'stamping' using the PBA robot
SD agar
Ampicillin and tetracycline [see SOP GLS/07]
Hybond N paper # [Amersham RPN119N]
Fine forceps
Methanol
Absolute alcohol
70% alcohol in wash-bottle
96-pin 'hedgehog'
Sterile 25ml pipettes [Sterilin 40125]
Sterile 5ml pipettes [Falcon 7543]
Sterile 50ml Falcon tubes [Falcon 2097]
Sterile 'universal' tubes [Sterilin 128A]
Sterile blue plastic loops [Nunc 254437]
Sterile TE [see SOP GLS/07]
Gloves (Microtouch)

GLOVES ARE WORN FOR ALL PROCEDURES


Pooling Scheme

All YAC libraries at the RC are now pooled and issued according to the same scheme.
The library is divided into stacks of 8 (ICRF, Lander mouse YAC) or 9 (ICI) plates.
A pool of these 8 or 9 plates becomes a primary pool. Thus ICI has 40 primary pools. ICRF 27 and the Lander mouse YACs 50 primary pools. Once a user has identified a positive primary pool they will receive a secondary pool from us. The secondary pools consist of plate pools from all plates in their positive primary pools, as well as pooled rows and columns from all plates in the pool (the corresponding rows and columns from each plate in the primary pool are combined to construct 20 pools). The user receives a total of 28 or 29 DNA pools, depending on the library (ICRF/Lander mouse or ICI). At least three of these pools should be positive in their PCR screening: one to determine the plate containing the positive clone, and two to determine the plate co-ordinates. All the pools are issued as DNA in agarose 'blobs' - before these blobs can be made the cells have to be collected.
(A diagrammatic representation of the screening method is shown at the end of the SOP)


Pouring the agar plates

Microwave the 500ml bottles of SD agar. Make sure that they do not spill over in the microwave. The melted agar can be stored in a hybridising oven at 65oC until ready to be poured. Add 500ml of ampicillin and 1.25ml of tetracycline to each bottle, once the agar has cooled down, just before pouring.
One 500ml bottle will pour approx. 10 round petri dishes or 14 Hybaid colony picking plates.
Only open the sterile plates within the open fronted safety cabinet (sterile hood).
Pour the agar gently into the plates.
Use a sterile loop to push any air bubbles to the side of the dish.
Allow the agar to set, and also to dry after setting before replacing the lid. (If the lid is replaced too early a lot of condensation is created on the agar. Thus when the yeast grows it is very wet, and this can present problems).
Store the plates, wrapped in clingfilm, upside-down, in the fridge until required.

Laying on the Hybond N paper

Working in the YAC laminar flow cabinet, the 'hood', lay the Hybond N filters on the agar. Wear gloves. Sterilise the flat ended, blue handled tweezers by dipping them in 100% alcohol and flaming them. REMEMBER TO KEEP THE ALCOHOL AND NAKED FLAME AT OPPOSITE CORNERS OF THE HOOD. As the hood floor should have been sprayed with 70% alcohol and wiped, it is safe to lay the tweezers on this floor.
Use the sterile flamed blue-handled tweezers to lift each membrane and place on the surface of the agar in the Hybond colony picking plate or round petri dish. The membrane must be laid absolutely flat, with no air bubbles underneath. (The corners will turn up when placed in the petri dishes - this does not matter as there will still be room on the agar to stamp the plate.)


Stamping the plates by hand (using petri dishes)

Thaw out your YAC working copy plates
Turn each petri dish upside down and label the plate number at the top of the plate.

You will need three agar plates for each YAC 96-well plate.

First agar plate

=

collecting the plate pools

Second agar plate

=

collecting all the rows

Third agar plate

=

collecting all the columns

Sterilise the hedgehog by placing in the methanol bath and flaming.
REMEMBER TO KEEP THE METHANOL AND NAKED FLAME AT OPPOSITE CORNERS OF THE HOOD. Allow the hedgehog to cool slightly. Place the source plate with well A1 at the top left, and place the three agar plates, with the correct plate number at the top of the plate. (Even though the number will be a mirror image as it was written on the plate upside down - it should be checked before you stamp out).

Place the hedgehog into the source plate, and then transfer it to the first agar plate and allow the pins to gently sit on the surface of the mambrane. Repeat this for the other two plates.

Place the hedgehog in a bath of sterile water, stamp dry onto kitchen towel, place in the methanol bath and then flame as before. Throw away the kitchen towel.

You are now ready to stamp the next plate.


Stamping the plates using the PBA robot (using agar in colony picking plates)

Modify the procedure given in GLI/1998/3/11. So that instead of replicating the source plate into a liquid 96-well plate, you will be replicating onto the agar plate.


Incubating the agar plates

Incubate the agar plates, wrapped in cling film and upside down, for 2-3 days at 30oC.
When the plates have grown they can be stored at 4oC if necessary before the cell pools are collected.


Collecting the plate pools.

Work in the YAC hood which has been cleaned before use, as normal.
Collect the first set of agar plates from the 8 or 9 plates in the stack (stack = primary pool) and place in the hood.
Label 8 (or 9) sterile universals with the correct plate numbers.
Using a sterile 25ml pipette add 15ml sterile TE to each agar plate.
For each agar plate, use a fresh sterile 25ml pipette and very gently move the TE around until all the cells are in suspension. Be careful not to be so vigorous that you collect agar as well. Use the pipette to transfer the 15ml of cell suspension to the correctly labelled universal. Remember to use a new pipette for each plate.
At the end of the stack, you will have 8 (or 9) universals with cell pools in them.
Using a fresh sterile 5ml pipette for each universal, remove 5ml of cell suspension into a single 50ml sterile Falcon tube. This tube will end up with 40ml (or 45ml) of cell suspension and will be the primary cell pool. It should be labelled appropriately (each library differs in its numbering of primary pools. See the team leader for details of the particular library that is being worked on)
Store the cell pools, in clearly labelled and dated racks at 4oC.


Collecting the super-rows

Because we are going to collect the rows from all 8 (or 9) plates and pool them together, we call these super-rows.
This work is done on the bench in the lab. The bench should be thoroughly cleaned with 70% alcohol before use.
Label 20 universal tubes with primary pool number and -1 to -20.
Fill tubes 1-8 with 15ml sterile TE using a sterile 25ml pipette. These will be used for the super-rows.
Fill tubes 9-20 with 10ml sterile TE using a sterile 25ml pipette. These will be used for the super-columns.
Take the second set of 8 (or 9) agar plates

Make sure the plates are all orientated correctly, with the PLATE NUMBER AT THE TOP.

Use a sterile blue loop to gently drag across all row A in plate 1 and lift the cells collected in the loop into the TE in the universal labelled xx-1.
Place this plate that you have already collected to a new position on the bench. (i.e. you want to be able to keep a clear mental image of the plates you have already collected from and the ones yet to be done)
Without changing the blue loop collect all row A from plate 2 and place it in the universal xx-1.
Do this for all the plates.
At the end you will have all row A, from 8 (or 9) plates, all together in the universal xx-1.
Put the lid on the universal and move it to a different rack.
Now open the lid of universal xx-2 and USING A NEW STERILE LOOP collect all the row Bs from the 8 (or 9) plates.
Continue, in this way until you have collected all the row Hs into tube xx-8.
Your agar plates should now have all their rows swiped off, and can be discarded.


Collecting the super-columns

Take the third set of 8 (or 9) agar plates

Make sure the plates are all orientated correctly, with the PLATE NUMBER AT THE TOP.

Use a sterile blue loop to gently drag down all column 1 in plate 1 and lift the cells collected in the loop into the TE in the universal labelled xx-9.
Place this plate that you have already collected to a new position on the bench. (i.e. you want to be able to keep a clear mental image of the plates you have already collected from and the ones yet to be done)
Without changing the blue loop collect all column 1 from plate 2 and place it in the universal xx-9.
Do this for all the plates.
At the end you will have all column 1, from 8 (or 9) plates, all together in the universal xx-9.
Put the lid on the universal and move it to a different rack.
Now open the lid of universal xx-10 and USING A NEW STERILE LOOP collect all the column 2s from the 8 (or 9) plates.
Continue, in this way until you have collected all the column 12s into tube xx-20.
Your agar plates should now have all their columns swiped off, and can be discarded.

All the universals of cell pools can be stored at 4oC until they are needed for spheroplasting. In our experience these cell pools still produce good DNA agarose blobs after 1 years storage at 4oC (they have even been used satisfactorily after 18 months storage)

PS. Another name for stacks is blocks. The Lander mouse YAC library is divided into 50 blocks - labelled B1 - B50 (B = blocks)


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

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