1 Fix screening 9x9mm gridded template to photographic light box : masking tape works well
2 Align autoradiographic film over template and fix with tape
Template:
3 Use permanent marker pen to grid-out colony area on film
4 Score positive colonies on record sheet (as below)
5 It is advisable to set up a spreadsheet using formula to extract individual plate results from the composite record sheet. This simplifies identification of wells in each plate for re-gridding.
1 Thaw copy set of colony plates
2 Label sufficient plates for re-gridding with unique identifier and date
3 Add 100 microlitres LB-amp broth
4 Tape insert from 8x12 tip box over plate for re-gridding This stabilises tips used to transfer culture supernatant from the copy set of colony plate and allows the easy remove of tips when plate complete: see below
5 Place sterile yellow tips in wells to be transfered: one row at a time to prevent contamination and confusion !!!!!
6 Gently tap tips in the well supernatant to encourage capilary uptake until ~5-10 microlitres in tip
7 Transfer tips to re-grid plate Note well position in re-gridded plate where each transferred row finishes: this allows transfer to be checked ie 8 wells transfered from row of copy set of colony plate = 8 wells in re-gridded plate
8 When re-grid plate completed carefully insert multichannel pipette:- Expel contents of tips Withdraw tips Discard tips in suitable waste container
9 Complete all well transfers as in step 8
10 Incubate plates at 37°C until growth visible in the majority of wells in the re-gridded plates Usually 4-6hr:
11 Add 50 microlitres 45% glycerol/LB broth to each well 12 Store plates at _80°C until required for sequencing
[ampicillin] = 50 micrograms/ml
LB BROTH (PER LITRE)