Colony Hybridization
- Transfer 100 colonies using a sterile toothpick in a grid pattern to duplicate LB/Carb 10 cm plates one of which contains a PALL-BIODYNE MEMBRANE (ICN Pharmaceuticals). Grow O/N @ 37°C.
- Treat the filters for 15 minutes by incubation on a 0.5 N NaOH saturated Whatman filter in a glass dish; 2X 1 minute with 0.5 M Tris pHÂ 7.6; and once for 15 minutes with standard renaturation solution. Bake 2 hours at 80°C in vacuum oven.
- For oligonucleotide probes, prehybridize 1 hour in 6X NET (1X=0.15 M NaCl, 15 mM Tris HCl pH 8.0, 1 mM EDTA), 10X Denhardt's, 0.1% SDS. Add 2,000,000 cpm/filter 32P-labelled olig°and hybridize O/N at 37°C. Note: for longer probes the stringency should be increased based upon the Tm of the probe and template.
- For oligonucleotide probes wash with 6X SSC/0.1%SDS 10 minutes at R.T.
- Repeat for 10 minutes at 37°C with the same prewarmed wash.
- Autoradiogram 1-4 hours.
- Repeat washes at 50°C, 55°C, 60°C, 65°C as needed and reautoradiogram until negative controls are negative.
- Grow 10 ml overnight of 2 positives.
- Standard Miniprep.
- Retransform with 50 ng of miniprep DNA and repeat screen on 30 colonies.
- Repeat steps 5-7 1X.
- Grow 20 ml minipreps of 2 final positive clones and check mutagenesis by sequencing.
Solutions:
- 1. Running Buffer:
8 ml Formamide
10 ul NaOH
10 mg bromophenol blue
2 ml H2O
- 10X TBE
1 M Tris (121.1 g)
51.3 g boric acid /liter pH=8.8
3.72 g EDTA
- 10X Hybridization Buffer
50 mM Tris pH 7.4
50 mM NaCl
1 mM EDTA
- 20X NET
M NaCl
0.3 M Tris pH 8.0
20 mM EDTA
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