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LABORATORY PROCEDURES FOR GENOMIC LIBRARIES

REFERENCE NO: GLI/1998/4/1


TITLE: ISSUING PAC POOL AND CLONE REQUESTS



PAC POOL REQUESTS

All the PAC pools are stored frozen at -70oC, as cell pools, and need to be dispatched to the user on dry ice in polystyrene boxes.

The orders therefore have to be assembled and packed on dry ice first thing in the morning, and taken to the office, so that the carrier can be contacted early in the day to come and collect them.

Materials

Polystyrene box
Dry ice
Parcel tape
Appropriate labels


Assembling the orders:

Primary Pools

These consist of bags with 21 tubes inside. The primary pools are stored in the -70oC freezer L49, bottom shelf. On the shelf is a blue rack, with holes labelled A to U. It is important to check that each bag contains 21 tubes, labelled with each of the 21 letters from A to U. So working rapidly, as the tubes must not thaw, place the tubes in their labelled holes in the rack. When you have ascertained that the correct tubes are present, place them back in the plastic bag and place them in a polystyrene box with dry ice. It is important to ensure that there is dry ice both below and above the bag. Fill the box to the top with the dry ice.

On the top of the dry ice, protected by a plastic bag, place a blue instruction sheet with (a) highlighted together with a blank request form. Place on the box lid a 'Freeze on Arrival' sticker (printed out, and stored, in the large blue PAC handouts lever arch file).
Tape the lid of the box on with parcel tape.
Score the record sheet on the wall thus keeping an up to date stock record of our primary pools.


Secondary Pools

The secondary pools, for each primary pool, have been aliquotted into 96-well plates and are stored in the -70oC freezer L52 (all shelves, labelled) or L49 (pool U). The requested plates are placed in dry ice in a polystyrene box, as above.
On top of the dry ice, protected within a plastic bag, place a blue instruction sheet with (b) highlighted together with the appropriate pink sheets for the pools requested
(these sheets are essential for the user to identify the positive plate pools) and a blank request form.
Package the box as above.
Score the record sheet on the wall thus keeping an up to date stock record of our secondary pools.


Individual Plates

Currently we have no robotics enabling us to provide users with rows and columns. Therefore, once a positive plate has been identified the user receives a copy of that plate, so that they can make their own rows and columns.


Materials required:

Genetix 384-well plates
LB broth + kanamycin
384-well 'hedgehog'
Bunsen burner
methanol in large petri dish
disposable plastic tray
autoclaved milli-Q water

Fill the plates with the LB broth using the BioRobotics Colony Picker (or the Genetix QFill)
Incubate a spare plate overnight at 37oC to check for contamination. Look at this plate next day to confirm it is free from contamination before replicating the PAC plates.
Remove the required PAC plates from the working copy freezer and thaw.
Work in the laminar flow hood. Spray clean with 70% alcohol before use
Wear gloves throughout
If required, the 384-well 'hedgehog' can be sonicated in the same way as the PBA Gridder tool.
Place the burner on the RHS in the hood
Place the methanol dish on the LHS
To flame the 'hedgehog' dip in the methanol bath, REPLACE the lid on this methanol bath, and only then flame the 'hedgehog'. It is essential to keep the methanol covered and as far apart from the burner as possible.
Leave the 'hedgehog' on its side to cool down
Place the source plate and the correctly labelled media-filled plate in front of you, side by side, ensuring that both plates are orientated correctly with A1 at the top LHS corner. Lift off the lids.
Place the 'hedgehog' into the source plate, holding the hedgehog firmly and pressing down into the frozen wells moving the pins slightly from side to side. Replace the source plate onto the dry ice.
Transfer the 'hedgehog' to the copy plate, and move the pins from side to side again to inoculate this plate.
[NB: when aligning the 384-well 'hedgehog' into a plate just look at the front row of pins, align them correctly, and the rest of the pins will also be in the right orientation.]
Place the 'hedgehog' into the plastic tray filled with milli-Q water to wash off the bulk of the cells.
Stamp the 'hedgehog' onto paper towels to partially dry it; then follow the methanol bath and flaming procedure described above.
Whilst the 'hedgehog' is cooling replace the lids on the plates you have just replicated and get the next set of plates ready.
When all the plates required have been replicated, place the source plates back into the freezer.
Wrap the copy plates in cling film and incubate at 37oC overnight.

Score the record sheet on the wall thus keeping an up to date record of the numbers of times that a plate has been thawed.

Next day check that the plates have grown well. The plates will then need to be frozen at -70oC before dispatching to the user.
The frozen plates should be packed in dry ice, as described for the pools. The information that the user requires is called 'Individual PAC clones', printed on orange paper together with the information sheet on DNA preparation. The information packs are in the filing cabinet.


Dispatching the frozen samples

At the moment we use three different carriers, depending on where the order is to be sent:

Cambridge,

hand delivered by the Lab Aid, if possible

UK

Europe

America

The office will phone up the companies concerned - but give them plenty of time to do this by forewarning them that parcels will brought down, and taking the parcels as early in the day as possible.

UK

This is straightforward. After packing, the polystyrene box has is taken to the office together with a adequate form. On the plastic bag label you have to complete the address of the user, and tick the next day delivery box before 12 noon. Both the plastic bag label and the forms are stored in the filing cabinet drawer. Also place a "freeze on arrival" sticker (printed out, and stored, in the large blue PAC handouts lever arch file) on the top of the box.

NB: the form has space for sending out to six different addresses, so you do not need individual forms if you are sending more than one parcel.

EUROPE

This is not straightforward. It is important that the parcels are taken to the office before 10am. However package the box in the normal way, take to the office and all the extensive forms that are required are there. The weight of dry ice has to be entered, for the standard size polystyrene box this is 3 Kg. The box also requires a class 9 diamond shaped sticker. Also place a "freeze on arrival" sticker (printed out, and stored, in the large blue PAC handouts lever arch file) on the top of the box.

America

We are unlikely to be sending many PAC requests to the USA but if we do the procedure is the same as for Europe with all the forms in the office.


PAC CLONE REQUESTS

Materials

LB agar + kanamycin poured as slopes or colony picking plates
Sterile yellow stackpack tips


Working in the plate pouring hood, wearing gloves, add the kanamycin (500Ál of 25mg/ml stock to the 500ml of melted LB agar).
Pour out the slopes or colony plates exactly as the method for SD agar described in
GLI/1998/3/2.
Before streaking the clones check that none of them are on the list of non-recombinants - do not send these clones.
Take out the required PAC plate, from the working copy freezer. Keep frozen, by placing the plate on dry ice.
Work in the laminar flow hood in the lab.
Use a sterile yellow tip to go into the desired frozen PAC well, and then carefully transfer and gently streak onto the agar. It is not necessary to remove a large amount of the frozen clone; very little is required to grow well.
Replace the PAC plate in the freezer before it thaws.
Incubate the clones overnight at 37oC.
Check that the clone has grown before posting
The clones are posted out in exactly the same way as the YAC clones; at RT. together with a new blank request form, growth conditions slip, and DNA isolation information.


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

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