Overgo Labeling Date ____/___/____

Species Filters _______________________________

Labeling Reaction:

(Reaction sizes can vary depending on how much you plan to add to the filters and how many filters you have. Determine which size reaction volume you will need and select from the Reaction Size table below.)

· Add of overgo working solution (2mM concentration) in a PCR tube and seal with a cap (see below for appropriate amount).

· Place in a thermocycler (BioRad 170-9703) and run a program for 80°C for 5 minutes, 37°C for 10 minutes, and 4°C ¥. Let oligos run at least 2 minutes at 4°C but no longer than 15 minutes

· Add the OLB, BSA and water to a tube on ice while waiting for the probes to incubate. As soon as the incubation is complete and the thermocycler drops to 5°C, add the isotopes and Klenow (NEB M0210S). Klenow comes with different activity concentrations so dilution with the appropriate amount of water is necessary.

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

For 1 full rxn. Add directly For multiple reactions mix in

to PCR tube 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 ____ ml = ____ml Klenow

· Mix well by drawing up and down with the pipet after addition of Klenow (avoid bubbles)

· For multiple reactions aliquot master 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 ____________

· Label ____ Nick columns (Amersham 17-0855-02) (max volume 100ml) and 1.5ml centrifuge collecting tubes, and equilibrate with 3ml of TE

· Add enough TE to the anchor probe to make a total of 50ml

· Take 1ul from the Anchor reaction and 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

· Throw out columns in radioactive dry waste

· Take another 1ml of the anchor probe and 1ml of test probe and add to separate scintillation vials

· Count vials for 0.5 minutes (2^nd Anchor vial should be greater than 10,000 cpm)

· 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°

· Unused probe can be stored at 4°C for later use up to one week.

Scintillation counts:

Count (dpm or cpm) Percent Incorporation

Anchor before column __________ _________

Anchor after column __________ _________

Probes after column __________ _________

Folder Name ___________________ Exposure Time ________hours

File name ___________________

Folder Name ___________________ Exposure Time ________hours

File name ___________________

Comments: ____________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________