Growth of Agrobacterium Transformation-competent Clones
- Grow bacteria in 1/2 strength M9 mediun (0.5X M9, 0.1% MSG, 0.2% glucose, 0.5% glycerol, 0.5 mM MgSO4, 60 ug/ml gentamicin sulfate). Inoculate and let bacteria grow for about one week in the microtiter dishes (room temperature).
Lysis of Agrobacterium Transformation-competent Clones
- Pellet cells using micotiter-dish swing in Sorvall RT600B centrifuge (3500 rpm, 12 min, 4C). Stack two microtiter-dishes per swing. Don't be alarmed if plates crack; well integrity should be maintained. Vacuum off supernatant with a 12-well suction apparatus.
- Add 100 ul Suspension buffer (50 mM glucose, 25 mM Tris (pH8), 10 mM EDTA, 1 mg/ml lysozyme, 100 ug/ml proteinase K). Mix cells carefully on vortexer.
- Add 100 ul Lysing mix (0.2 N NaOH, 1% sarcosyl). Incubate for at least 10 minutes or until complete cell lysis.
- Add 100 ul 3/5M Potassium Acetate (Possibly skip here if samples blotted immediately).
- Place microtiter-dishes at 4C until ready to blot.
Transfer of DNA to Nylon Membranes
- Use Bio-Rad Dot Blot apparatus. Use Bio-Rad Zeta-probe membranes cut to fit apparatus. Soak cut Zeta-probe membranes in water, then fit into vacuum apparatus.
- Heat microtiter dish for 6-8 minutes at 99C (or place in microwave for 30 seconds).
- Add 200 ul water to wells of apparatus. Apply suction till water level clears wells and stop suction.
- Apply 200 ul cell lysates with a 12-channel (or 96-channel?) pipetter. Apply suction again.
- Add 200 ul 0.4 N NaOH once most of the lysate has passed through the filter. Continue suction until all liquid (or most of it) has passed through the membrane.
- Remove excess liquid.
- Turn suction off and remove filter.
- Wash briefly in 2X SSC (float on liquid or place on soaked filter paper).
- Air dry filter, and vacuum bake at 80C for 30 minutes.
Hybridization of Nylon Filters with DNA Probes
- Label probes using the random hexamer-primed labelling method.
- Hybridize filters with cleaned probe in hybridization buffer (0.25M Na/P buffer, 7% SDS, 1mM EDTA, 1% BSA) overnight at 65C. [1 M Na/P = 134 g/L Na2HPO4*7H2O, 4 ml/L 85% H3PO4]
- Wash filters sequentially in 2X SSC, 0.1% SDS; 0.5X SSC, 0.1% SDS; and 0.1X SSC, 0.1% SDS at 65C.
- Wrap filter wet in saran wrap and expose to X-ray film (plus intensifying screen at -80C).
Stripping Filters of DNA Probes
- After hybridization and exposure, try not to let the filters dry out. Place filters in 0.1X SSC, 0.5% SDS and heat two times at 95C for 20 minutes. Use as soon as possible for next hybridization experiment.
- Check stripped filter by exposing to X-ray film overnight.
Analyzing Results
- The 225 filters were oriented in a 15X15 format. By analyzing positive signals in one orientation (15 vertical pools or 15 horizontal pools), a signal in a similar location is looked for in the other orientation. Once an identical positive signal is identified in both orientations, the clone can be isolated from the designated location within the master library. Alternatively, 1 out of 15 colonies from a pooled microtiter dish well will yield a positive clone. If the label is still of high-energy, then colony hybridizations may be done to identify the positive clone.
