Secondary Screen Protocol

•Turn on hybridization oven (Fisher isotemp 13 247 31) and heat to 58°C. If this is the first use of the filters, skip the following step and go right to placing the filters in to the hybridization bottles.

· If filters have already been used and need to be stripped, prepare stripping buffer and heat it close to boiling in a microwave (4.5 minutes on high for 1L). Place the filters into a Clear Magazine Snap Case (14”x 8 5/8”x 3 7/8” The Container Store 584070) and pour the stripping buffer over the filters. Place the container into a a 5 piece Pyrex Portable with heated hot packs (microwave for 4 minutes ahead of time) for insulation. Shake on a rotating platform for 15 minutes. Pour off the stripping buffer when done shaking.

· Place filters into 150mm hybridization tubes (Fisher 13 247 150)

· Add 5ml Church buffer (see reagents) to each tube. If the filters have been used previously, incubation time needs to be only 20 minutes. If this is the first use, the filters need to be incubated for at least 3 hours and then the church buffer poured off and replaced with fresh 5ml of buffer.

· Incubate in oven while labeling primers

· Set up labeling reaction for overgo primers (see overgo worksheet)

· Pour out the pre-hyb church buffer and add 5ml fresh church buffer

· Add denatured probes down the center of the tubes and incubate overnight at 58°C. Typically 10ml of anchor and 50ml of probes.

· Take the tubes out of the oven in the morning and pour the hybridization buffer into liquid waste

· Add 20ml of 1.5X SSC/ 0.1% SDS to each of the tubes to wash. Place back in the hybridization oven at 58°C for 30 minutes

· Pour off the first wash and wash again for 30 minutes with 20ml 1.0x SSC at 58°C

• Wrap with saran and expose to phosphor imaging screens. Cut a large piece of saran and lay it flat on the desktop. The phosphor imagers will fit six filters with about a 1/4 inch between them. Place the filters face down on the saran, placing the ones you want on the right side of the exposure on the left side and vice versa. Arrows should be pointing to the top of the cassette for exposure.

· Cut a piece of blotting paper to fit the inside of the imaging cassette (Amersham RPN11643). Tape the covered filter face up on to the filter paper. Place the imaging screen (Amersham 63-0034-90) face down on to the covered filter and close the cassette for exposure.