BAC Fiber-FISH

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BAC Fiber-FISH

Prior to preparing slides:

1         Label appropriate probes with biotin and dig (see Probe Labelling protocol).

2.         Mini-prep BAC DNA (Alkaline lysis method; use 20 µl water for resuspension).

3.         Silanize 22x22 coverslips by dipping in Sigmacote for 10 min, then air dry.

Slide preparation:

4.         Prepare wet-chamber at 37°C, turn slide warmer up to 60°C.

5.         Dilute BAC DNA (w/ cut P20 pipet tips) to appropriate level (We like to dilute 1 µl BAC into 9 µl water).  Add all 10 µl      of diluted BAC to Poly-Prep slide (Sigma #P0425).

6.         Add 15 µl of FISH lysis buffer* to BAC drop.  Allow drop to spread.  Let this sit at room temperature for ~5 min.  Add water to the slide if it dries.

7.         Gently place (“drop”) a silanized coverslip directly over the liquid (lower the slide with forcepts to avoid bubbles).

8.         Transfer slides to slide warmer.  Allow slides to “bake” for 15 min.  At this point one should see the liquid begin to recede.

9.         Place slides in 3:1 (EtOH: Glacial Acetic Acid), wait 1 min.  Gently shake slide to promote removal of the coverslip.  Once coverslip falls off, transfer slides to new container of 3:1 and incubate for 1:30.  Transfer slides back to slide warmer for an additional 15 min.

10.       Add probe, denature, and detect as in Nuclear Fiber-FISH protocol.

  *FISH lysis buffer :

            2% Sarkosyl

            0.25% SDS

            50 mM Tris (pH 7.4)

            50 mM EDTA (pH 8.0)