Colony Blotting with 32P

Colony Blotting with ³²P
This is by far the best method for use with³²P - 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.

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

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

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