Colony Blotting with 32P
Categorized: Bacterial Genetics
This is by far the best method for use with 32P – works every time and the pencil marks develop on the film for easy orientation of the colony lifts!
- Grow colonies overnight to 1-3 mm in diameter.
- Blot onto Whatman 541 filter paper. Label top side on front with a pencil. Air dry.
- In a large glass tray saturate several sheets of Whatman 3MM paper with 0.5 N NaOH. Lay filters colony side up on the saturated paper and leave for 5 minutes. Colonies will turn slimy as they lyse.
- Transfer filters to a tray with 1 M Tris pH 7.4 and leave for 5 minutes with occasional agitation.
- Transfer the filters to a tray with 2X SSC and leave for 5 minutes with occasional agitation.
- Transfer filters to a tray with 95% ethanol. Agitate for 5 minutes. Ethanol will become cloudy. Replace ethanol if doing many filters.
- Air dry. Do not bake.
- Place filters in a plastic box with 500 ml 5X SSC and 0.2% SDS. Incubate with shaking at 65C for 30 minutes to remove cell debris.
- Drain filters and place in a seal-a-meal bag. Add hybridization buffer.
0.25 M NaHPO4 6.25 ml 1 N NaHPO4 1 mM EDTA 0.05 ml 0.5 N EDTA formamide (50%) 12.5 ml 7% SDS 1.75 g water 6.2 ml
- Prehybridize for 5-10 minutes at 37C. Add denatured probe and hybridize 24 hours at 37C.
- Wash filters for 5 minutes at RT with 2X SSC.
- Wash filters for 2 hours at 37C with 250 ml:
0.25 M NaHPO4 75 ml 1 N NaHPO4 1 mM EDTA 0.05 ml 0.5 N EDTA 2% SDS 50 ml 10% SDS water 124.5 ml
- Wash for 2 hours with:
0.15 M NaHPO4 37.5 ml 1 N NaHPO4 1 mM EDTA 0.05 ml 0.5 N EDTA 1% SDS 25 ml 10%SDS Water 187 ml
- Drain and air dry. Tape filters to a supporting sheet, cover with Saran wrap and autoradiograph.
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