DNA purification

Run PFGE as in other protocol.
Excise bands (use unrun plugs similarly to make reference DNA) and place in tubes with 1 ml TE. Incubate room temperature 30 minutes.
Replace TE with fresh TE and incubate 30 minutes.
Replace TE with 250 microliters 1X restriction enzyme buffer and incubate 30 minutes.
Replace with new buffer and incubate 30 minutes.
Replace with new buffer plus 100 micrograms/ml BSA.
Add 30-50 units restriction enzyme. Incubate overnight at appropriate temperature.
Add a boost of half the other amount of restriction enzyme in the morning and let incubate a few more hours.
Purify the digested DNA using a Qiagen gel purification kit.

Amplification

Round A
Combine in a PCR tube:
1 microliter 40 micromolar Primer A (5'-GTTTCCCAGTCACGATCNNNNNNNNN-3')
2 microliters 5X sequenase buffer
7 microliters purified DNA

Incubate 2 minutes 94 degrees C.
Hold at 8 degrees C for 2 minutes.
Add 5 microliters reaction mix.
8 minutes ramp to 37 degrees C.
8 minutes at 37 degrees C.
2 minutes 94 degrees C.
Hold at 8 degrees C for 2 minutes.
Add 0.3 microliters sequenase and 0.7 microliters sequenase dilution buffer
8 minutes ramp to 37 degrees C.
8 minutes at 37 degrees C.

Reaction mix (for 1 sample)
1 microliter 5X sequenase buffer
1.5 microliter 3 mM dNTPs
0.75 microliters 0.1 M DTT
1.5 microliters 500 micrograms/ml BSA
0.3 microliters 13 U/microliters sequenase

Dilute product to 50 microliters with 35 microliters TE.

Round B

15 microliters diluted Round A
10 microliters 10X Taq buffer
1 microliters 25 mM dNTPs
2.5 microliters 500 pm/microliter Primer B (5'-GTTTCCCAGTCACGATC-3')
1 microliter Taq
8 microliters 25 mM magnesium chloride
62.5 microliters water

92 degrees C 30 seconds
40 degrees C 30 seconds
50 degrees C 30 seconds
72 degrees C 1 minute
25 cycles

Labeling

15 microliters Round B
5 microliters 10X Taq buffer
0.5 microliters F-100X dNTPS
1 microliter 500 pm/microliter Primer B
3 microliters Cy dye
0.5 microliter Taq
4 microliters 25 mM magnesium chloride
21 microliters water

F-100X dNTPS (40 microliters)
10 microliters 25 mM dATP
10 microliters 25 mM dCTP
10 microliters 25 mM dGTP
5 microliters 25 mM dTTP
5 microliters TE

Same PCR as Round B, expect 2 minutes at 72 degrees C.

Mix probes in microcon 30 and add 400 microliters TE.
Spin 8 minutes 10000 rpm microcentrifuge.
Add 450 microliters TE.
Concentrate to 23.1 microliters (or whatever is appropriate for your array size). Collect and bring to 23.1 microliters.
Add:
8.1 microliters water
6 microliters 20X SSC
3 microliters 5 mg/ml yeast tRNA
0.9 microliters 10% SDS
Boil and hybridize overnight at 65 degrees.

Washes

2 minutes each, at room temperature.

wash 1
400 ml
20 ml 20X SSC
1.2 ml 10% SDS
Let coverslip float off in this wash. Let arrays sit in this solution until all coverslips are off, then start 2 minute agitation. Transfer each slide individually to wash 2 to minimize SDS carryover.

wash 2
400 ml
4 ml 20X SSC

wash 3
400 ml
1 ml 20X SSC