M. Fero 4/6/2006
(courtesy of Katie Rudd, Trask lab)
20 ng BAC DNA clone grown o.n. in 5 mL LB + chloramphenicol
Organics: MeOH, EtOH, formamide
Fixative (3:1 MeOH:glacial HOAc)
Biotin (or digoxigenin) nick-translation Kit (Invitrogen)
20x SSC (3 M NaCl, 0.3 M NaCitrate, pH 7.0)
CoT DNA 1 μg/μL (Invitrogen)
Glass slides, 18 mm glass slips
3 M NaOAc
Na Azide solution (e.g. 25% stock)
Nonfat dried milk
CHAPS or TWEEN detergent
Biotinylated goat anti-streptavidin (Vector)
Day 1: Prepare cells and DNA
- Prepare BAC DNA probe on Autogen machine (see separate protocol)
- Run 4-6 Autogen tubes per BAC.
- Hyb buffer: 50% formamide, 2x SSC, 10% dextran sulfate
- Cells: Feed control cells with fresh RPMI + 15% BSA.
Day 2: Check DNA and spots cells onto slides
- Resuspend DNA in 50 μL/autogen tube and pool in a 1.5 mL tube.
- Digest with Eco RI (5 μL DNA, + 1 μL enzyme, + 1.5 μL 10x buffer, + 7.5 μL H2O) 37ºC x 1-2 hrs.
- Run on 0.8% agarose gel (in 0.5x TBE gel + 0.5 μg/mL ethidium Br) x 2 hrs, to check DNA quality.
- Pellet cells and wash 1x in PBS.
- Optional: Treat cells with hyptonic solution.
- Pellet and resuspend in 3:1 MeOH:Glacial Acetic acid
- Cells can be kept at -20ºC in fixative, but if so they should be washed with fresh fix on the day of use.
- Place a wet paper towel on 37ºC hot plate.
- Wash slides by placing in 100% MeOH in Coplin jar at room temp.
- Wipe both sides with Kimwipe
- Breathe on slide to moisten surface
- Drop two spots of cells from a Pasteru pipette 6 - 12'' above slide.
- Place slide on moist paper towel on warmer for 1 min.
- Move to dry part of slide warmer and leave it here o.n. to age (or place in 65ºC oven x 1hr.)
Day 3: Label DNA and setup hybridization
- Setup nick-translation:
- 15 μL BAC DNA (~ 1 μg)
- 5 μL 10x dNTP mix
- 5 μL enzyme
- 25 μL H2O
- Incubate at 16ºC x2 hrs, then place on ice.
- Run a 5 μL aliquot on a 1% agarose gel to confirm fragmentation in the 200-600 bp. (If fragments are too large the DNA then return tube to 16ºC for 1 more hr.)
- Stop reaction with EDTA solution. (DO THIS BEFORE ADDING CoT!)
- Add 1 μL CoT DNA (1 μg)
- Ethanol precipitate by adding:
- 200 μL TE (final vol 250 μL)
- 25 μL 3 M NaOAc
- 550 μL EtOH
- Incubate at -20ºC ≥ 1 hr.
- Spin 5' to pellet DNA. Wash with 70% EtOH 2x to remove salts, air dry.
- Resuspend DNA pellet in 55 μL of Hyb buffer.
- Melt probe by incubating at 72ºC x15 min.
- Incubate at 37ºC x30 min. to allow CoT DNA to anneal to probe.
- Place probe on ice until ready to use.
Meanwhile in Coplin jars:
- Hydrate: 2x SSC x2-5 min.
(Optional, to reduce cytoplasm bkgd: 10 mg/mL RNAse (DNAse-free) in 2x SSC, 37ºC x30 min.)
- Denature in 2x SSC + Formamide (50% vol), 72ºC x2-5 min.
- Dehydrate: Ice cold 70% EtOH, 85% EtOH, 95% EtOH, 100% EtOH, each 5 min.
(Denaturing solution and EtOH washes can be reused if kept in bottles).
- Dry off back of slide with Kimwipe, and air dry by propping on one end.
- Add 10 μL of probe to a region of slide with a spot of cells and cover with a 18 mm glass slip.
- Seal edges with rubber cement using a 5 mL syringe. (Optional warm cement at 37ºC to reduce viscosity)
- Place slides in a humid chamber o.n. at 37ºC.
Day 4: Washes and probe detection.
- Place 6 Coplin jars in 42ºC bath: 3 jars with 2x SSC + Formamide (50% vol), and 3 jars with 2x SSC only.
- (Reagents can be reused if kept in screw capped bottles - note order of reagents as first buffer will be the most "dirty")
- Warm blocking agent to room temperature (4x SSC, 5% non-fat dry milk, 0.02% Na Azide).
- Remove slides from humid chamber. Remove rubber cement. Gently slide off glass slips (dip in PBS if necessary).
- Wash slides:
- 2x SSC + Formamide (50% vol). 42ºC, x3.
- 2x SSC x5 min. 42ºC, x3.
- Blocking step:
- Wipe off back of slide.
- Add 100 μL block solution (4x SSC, 5% milk, 0.1% Tween-20) to the slide in 2 spots. (To store blocking reagent add 0.025% NaAzide and refrigerate at 4ºC)
- Cover with a large glass slip for 5'.
- Gently slide off slip.
- Labeling step:
- Add 100 μL of antibody (e.g. 1/400 avidin-FITC, Vector labs) diluted in blocking solution,
- Cover with large slip. Incubate r.t. x 30 min.
- Wash step:
- Gently slide off cover slip.
- Wash in 2x SSC + 0.005% CHAPS at room temp. for 5 min. (Repeat 3x)
- View on fluorescent microscope for signal. BAC probes probably do not need further signal amplification.
- If a second probe is being used on the same specimen then repeat the Block - Labeling - Wash steps for each (e.g. with anti-digioxigenin Texas Red).
- If a small probe was used or if the signal is too dim then the signal can be amplified further with additional rounds of:
- Biotinylated goat anti-streptavidin antibody (Vector) 0.5 mg/mL. Dilute 1/100 in