Secondary Screen Worksheet Date ____/___/____

Filters _______________________________________

Turn on hyb oven and heat to 58°C

Place dry filter into hyb tubes and label Score panel_______

Add 5ml Church buffer to each tube

Incubate in oven while labeling primers

Column Filter

1 _________ _________

2 _________ _________

3 _________ _________

4 _________ _________

5 _________ _________

6 _________ _________

7 _________ _________

8 _________ _________

9 _________ _________

10 _________ _________

11 _________ _________

12 _________ _________

Row

1 _________ _________

2 _________ _________

3 _________ _________

4 _________ _________

5 _________ _________

6 _________ _________

7 _________ _________

8 _________ _________

Labeling Reaction:

· Add the appropriate amount (see below) of 2mM overgo primers in a PCR tube and seal with a cap

· Place in Bio-Rad MyCycler and run the “oligos” program (80°C for 5 minutes, 37°C for 10 minutes, and 4°C forever)

· Let oligos run at least 2 minutes at 4°C

Reaction size ___ 5ml ___ 8ml ___full 10ml ___15ml Overgo primers 2.75ml 4.4ml 5.5ml 8.25ml

Master Mix 2.25ml 3.6ml 4.5ml 6.75ml

· Make master mix by adding in the following order:

For 1 full rxn. Add For multiple reactions mix

Directly to PCR tube in separate tube then aliquot

2 ml OLB ____rxns X 1.2 X ____ ml = ____ml OLB

0.5 ml BSA (2mg/ml) X 1.2 X ____ ml = ____ml BSA (2mg/ml)

Offset Klenow X 1.2 X ____ ml = ____ml dH₂O

0.5 ml ³²P-dATP X 1.2 X ____ ml = ____ml ³²P-dATP

0.5 ml ³²P-dCTP X 1.2 X ____ ml = ____ml ³²P-dCTP

1.0 unit Klenow X 1.2 X ____ units = ____ml Klenow

· Mix well by drawing up and down with the pipet after addition of Klenow

· For multiple reactions aliquot master (table above) mix to each of the probes (mix up and down ~5 – 10 times using a new tip for each reaction)

· Reseal tubes and place in a rack inside plexiglass box

· Let reaction go at least 1.5 hours at room temperature

Incubation start time ____________ Finish time ____________

· Turn on the heating block and set to 94°C by hitting one of the buttons

· Label ____ Nick columns and 1.5ml collecting tubes, and equilibrate with 3ml of TE

· Add enough TE to the anchor probe to make a total of 50ml and take 1ul to add to a scintillation vial.

· Add the reaction mixtures to the Nick columns

· Wash the Nick column with 400ml TE buffer and let drain

· Place the collecting tubes under the columns and elute probes with 400 ml TE

· Add 1ml of the anchor probe and 1ml of test probe to scintillation vials

· Count vials for 0.5 minutes

Scintillation counts: Count (dpm or cpm) Percent Incorporation

Anchor before column __________ _________

Anchor after column __________ _________

Probes after column __________ _________

· Denature probes for 5 minutes at 96°C

· Add ____ml of probes and ____ml of anchor down the center of the hybridization bottles and incubate overnight at 58°

· Wash and expose in the morning

Comments ______________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________

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