ISSUING cFUGU FILTER SETS AND CLONE REQUESTS

LABORATORY PROCEDURES FOR GENOMIC LIBRARIES

REFERENCE NO: GLI/1998/4/2

TITLE: ISSUING cFUGU FILTER SETS AND CLONE REQUESTS

Filter Requests

Take a set of cFugu filters, and ensure that the pack contains the correct number of filters and that it is a complete set.
Label the outside of the plastic bag containing the filters with the Req. No. (requisition number) assigned to it on the request form.
Paper clip a 'Genomic Clone Request - Hybridisation Only' request form to the original request form.
Take the filter set and the request form to the office and place in the out-tray.

Clone Requests

Check that the clone request forms have been fully completed before any clones are sent out. If the user has not filled in the sections 'probe(s) used' or 'chromosomal locations' then send the user a FAX requesting this information: the FAX should point out that the clones will be sent when this information is received.

Materials

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

Working in the E. coli hood, wearing gloves, add the kanamycin (600µl 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.

Make a list of all the clones requested in your lab. daybook/diary: this should be a record of the user, req.no., date in and date out.

Consult the Fugu data base to determine if any of the clones are known to contain a cosmid with no insert (ie; no Fugu DNA). Do not send these clones to the user, but identify them, and supply the explanation sheet explaining this.

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 the corresponding plates of the cFUGU library (gridding copy) out of the -70^oC freezer. Keep frozen, by placing the plate(s) on dry ice.

Use a sterile yellow tip to go into the desired frozen cFUGU 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 plates in the freezer before they thaw.

Incubate the clones overnight at 37^oC. 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.

Before sending out the clones the plates/agar should be examined to determine that all the clones have grown. If a clone has not grown check the record sheets for the Fugu library to see if that clone is absent. If the clone should be present, it will have to be restreaked, and the existing clones placed in a 4^oC fridge awaiting this clones' growth. If this clone is absent then send the explanation sheet to the user.

The clones are posted out in exactly the same way as the YAC clones; at RT. together with a new blank genomic clone request form and the information sheet called 'clones: Fugu cosmid library in E.coli'. The agar plates should have the edges sealed with parafilm, and then be wrapped individually in bubble wrap. The slopes should be placed in a plastic bag. Both the slopes and the plates should be identified with the Req. No. Take the clones and the request forms to the office and place in the out-tray.

Guidelines prepared for CABRI by HGMP, December 1998
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