Library Screen Worksheet date ________________

Species Filters_______________________________ Emory Grid _______

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

· Place in a thermocycler 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°

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 ____ ml = ____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 a plexiglass box

· Let reaction go at least 1.5 hours at room temperature

Incubation start time ____________ Finish time ____________

· Label ____ Nick columns and 1.5ml collecting tubes, and equilibrate with 3ml of TE (100mL max volume per column)

· 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.

Filter Cassette # Filter Cassette#

_______ _______ _______ _______

_______ _______ _______ _______

Exposure time _____hrs Exposure time _____hrs

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

Filter Cassette # Filter Cassette#

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