Hybridization Protocol for BAC Colony Filters

0.5 M Sodium Phosphate, pH7.2
134 g Na2HPO4.7H2O in 850 ml H2O
pH to 7.2 with 4 ml 85% H3PO4
adjust to 1 L with H2O

Hybridization solution (1% BSA, 1 mM EDTA, 7% SDS, 0.25M sodium phosphate)
500 ml 0.5 M sodium phosphate, pH7.2
10g BSA(Fraction V, Sigma)
70g SDS
2 ml 0.5 M EDTA, pH8.0
add H2O to make final 1000 ml

Prehybridization
Roll BAC filters and insert into a 300 ml hyb bottle. Add hyb solution, 30 ml per filter, and allow prehybridization at 65 degrees C for overnight when using new filters. Used filters may be prehybed for 3 hours and the probe added the same day.

Hybridization
Discard prehyb solution if using a new filter and add fresh hyb solution, 30 ml for 1-2 filters, 60 ml for 3-6 filters, and 100 ml for up to 10 filters (we normally only use up to 10 filters in one hyb bottle), continue prehyb at 65 C for another 1 hr before adding denatured probe. Incubate the probe hybridization for 16 to 18 hrs at 65 degrees C. The prehyb solution does not have to be changed for the used filters.

Washing
Remove hyb solution and rinse filters with 200 ml room temperature 1X SSC, 0.1% SDS. Discard the washing solution and transfer the filters to a large tub. Add 2L 65 C preheated 1X SSC, 0.1% SDS and continue washing at 65 C with gentle shaking for 1 hr. Further wash with 0.5X SSC, 0.1% SDS at 65 C if higher stringency is desired. Please be aware that you do need a little bit of background to help in identifying hit addresses and therefore we do not prefer washing too stringently. In most cases, one or two washes with 1X SSC, 0.1% SDS is sufficient.

Autoradiography
Mark the fields of the filters with radioactive ink. Place filters in plastic bags and expose to phosphoimager screens or X-Ray films for 6 hr to overnight.

Comments on Probe Preparation
The probes need to be vector sequence free to avoid high background signal. We use overgo probes for high-throughput library hybridization screening. The temperature for hybridization and washing is 60 C when overgo probes are used.

Stripping BAC colony filters
Wash filters for 10 min at room temp with gentle shaking in 100 mM NaOH, 10 mM EDTA, 0.1% SDS. If necessary, repeat this step until no signal is detected. Remove stripping solution by rinse with ddH20 and then wash in 5X SSC for 10 min. Repeat another wash in 5X SSC. The filters are now ready for another hyb or can be dried in 55C oven for 4 hr followed by storage at room temperature.