?

Hiro Hirai 's BAC-FISH Protocol

Overview

This protocol is a modification of the standard FISH protocol published by Hiro Hirai and Phil LoVerde in Parasitology Today (1995, 11(8) p 310-314) that has been optimised for use with BAC probes and should be read in conjunction with the published protocol.

This protocol uses the BIOPRIME reaction kit from GibcoBRL to prepare biotin-labelled BAC DNA which is detected using FITC-Avidin (Vector Labs, DCS grade). Reagents from other manufacturers may work equally well but have not been tested.

?

(1) Labeling of BAC clones

(a) take 10μl of standard stock solution of purified BAC clone and denature by boiling for 5 minutes, then snap cool in ice-water mix

(b) prepare 50 μl of labelling mixture as follows:

10μl denatured DNA 5μl 10x biotinylated dNTP stock (BIOPRIME reaction kit) 20μl 2.5x buffer solution (BIOPRIME reaction kit) 14μl ddH2 O 1μl Klenow enzyme (BIOPRIME reaction kit)

(c) incubate at 37o C for 3 hours

(d) stop reaction by heating to 65o C for 10 minutes

?

(2) Ethanol precipitation

?

(a) add together:

labelling mix (from 1.d) - 50μl 147μl ddH2 O 74μl 4M NaCl 2μl salmon sperm DNA (10μg/μl stock) 467μl cold (-20o C) ethanol

(b) keep at 4o C for 60 minutes

(c) centriufuge at 15,000 RPM at 4o C for 5 minutes

(d) remove supernatant, briefly dry pellet and then resuspend in 40 μl ultrapure formamide (Boehringer Mannheim or Intergene (Cat. No. S4117))

?

?

(3) Hybridization

(a) hybridization buffer:

15μl 20 x SSC 45μl 30% Dextran Sulphate

(b) hybridization mixture:

30μl hybridization buffer (from 3.a) 20μl labelled probe redissolved in formamide (from 2.d)

(c) denature the hybridization mixture at 72o C for 10 minutes

(d) denature the chromosome spread as folows:

0.05M NaOH in 2 x SSC - 4.5 minutes 70% ethanol - 5 minutes 99.5% ethanol - 5 minutes air dry

(e) apply the denatured probe to the chromosome spread, cover with parafilm (acting as a coverslip) and incubate in a humid chamber at 37o C for 12-16 hours

?

?

(4) Post-hybridisation treatment / detection

(a) wash:

40% formamide (standard grade) in 2 x SSC at 45o C for 10 minutes 2 x SSC at 45o C for 10 minutes 2 x SSC at room temperature for 10 minutes

(b) immersion and blocking:

BN buffer (0.1M NaHCO3 / 0.1% NP-40) or BI buffer (0.1M NaHCO3 / 0.1% IGEPAL CA-630 (Sigma I-3021 (as NP-40 may no longer be available))) for 5 minutes 5% non fat milk in BN or BI buffer for 10 minutes

(c) detection:

50μl of 5% non fat milk in BN or BI buffer containing 4μg FITC-Avidin (Vector Labs, DCS grade) for 60 minutes

(d) wash in BN or BI buffer, 2 x 10 minutes, with gentle shaking

(e) mount in antifade solution containing PI and DAPI as counterstains

?