Colony Blotting with 32^P

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!

1. Grow colonies overnight to 1-3 mm in diameter.
2. Blot onto Whatman 541 filter paper. Label top side on front with a pencil. Air dry.
3. 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.
4. Transfer filters to a tray with 1 M Tris pH 7.4 and leave for 5 minutes with occasional agitation.
5. Transfer the filters to a tray with 2X SSC and leave for 5 minutes with occasional agitation.
6. Transfer filters to a tray with 95% ethanol. Agitate for 5 minutes. Ethanol will become cloudy. Replace ethanol if doing many filters.
7. Air dry. Do not bake.
8. 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.
9. 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

10. Prehybridize for 5-10 minutes at 37C. Add denatured probe and hybridize 24 hours at 37C.
11. Wash filters for 5 minutes at RT with 2X SSC.
12. 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

13. 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

14. Drain and air dry. Tape filters to a supporting sheet, cover with Saran wrap and autoradiograph.