Mating Protocol | California State University, Northridge - Edubirdie

University:
California State University, Northridge
Course:
CHEM 101 | General Chemistry I
Academic year:

2004
Views:

300

Pages:

1
Author:

Charity Bullock

Mating Protocol Preliminary calculations: Want 250µl of OD600=10 per filter, or 2.5 OD's/filter for EACH E.coli strain. Want 500µl of 100µg/ml chla for each filter, or 50µg Chla/filter. 1. Have 500ml exponential ammonia grown Nostoc ready. Take chla reading and harvest enough for all matings. Resuspend to 100µg chla/ml and use 500µl per filter. 2. Inoculate 1/40th overnight culture of eacn E.coli strain into selective LB. Check OD600 and use when near 1. Calculate total OD600 units per culture. Centrifuge and resuspend to OD=10. Mix E. coli strains 1:1, to allow for 500µl per mating filter. Centrifuge and washtwice in LB. 3. Wash Millapore HATF nirocellulose filters in d water and place between Watman #1 filters. Autoclave in glass petri dish dry cycle( 20-20min). Place atop AA mops, ammonia, 0.5% LB mating plates. 4. Mix 500µl each Nostoc and E.coli from step 1&2 in a microfuge tube and spin briefly in microfuge. Pipet off all but ~200-300µl of supernatant and resuspend by pipetting up and down. Place atop mating filter (step 3) and spread. Allow for moisture to be absorbed/dried before parafilming. 5. Place in low light (bottom shelf CO2 incubator) overnight. Tansfer filter to AA mops ammonia and allow to recover in low light. When cells look healthy transfer filter to AA,mops ammonia, antibiotic plates, and place in high light

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