Hybridization

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Hybridization of the Probe to Chromosomes

1.     Remove the cover slips of the slides (stored in a -80°C freezer) with a razor blade and dry slides in an ethanol series (70%, 95%, 100% ethanol 5 min each at room temperature).  The slides should be dried several hrs before in situ hybridization.

2.     Prepare the hybridization mixture as follows:

        deionized formamide                                             10 µl

        20X SSC                                                                  2 µl

        Sheared salmon sperm DNA (10 mg/ml)                 2 µl

        Probe DNA                                                              1-2 µl

        H2O                                                              ? µl

        50% dextran sulfate                                                4 µl

        Total                                                                       20 µl

        Notes: Use H2O to adjust the total volume to 20 µl.  10 µl will be applied to each slide with a 18 ´ 18 mm cover slip.  Make sure the solution is well mixed because the 50% dextran sulfate is very sticky.

3.     Denature this mixture by placing at a 80°C heating block for 5 minutes and immediately chill the mixture on ice.  Spin down the solution.

4.     Add 150 µl of 70% formamide in 2X SSC solution on the dried slides and cover the slides with 22 ´ 40 mm cover slips.  Place slides on a plate (Metal or glass) in a 80°C hot oven for 1.5 min. (10 ml stock solution of 70% formamide in 2X SSC: 7 ml formamide, 1 ml 20X SSC, 2 ml H2O)

5.     Remove the cover slips by swing the slides and dip the slides immediately into an cold ethanol series (70%, 95%, and 100%, 5 min each at -20°C) and air dry the slides.

6.     Apply 10 µl hybridization mixture to each slide and cover with a 18 ´ 18 mm cover slip.  Seal the cover slip with rubber cement and place the slides in a wet chamber.

        Notes: You can use any kind of boxes with 2 layers of wet 3 mm Whatman paper on the bottom, use plastic bars or something else to hold the slides.

7.     Incubate the wet chamber at 37°C for minimum 6 hrs or overnight.

Notes:

a.     You may check the dried slides under a phase contrast microscope to make sure the metaphase cells are still there and the chromosomes are still in good shape (flat).  This is very important.  You will never get satisfactory results if the chromosomes become ugly after drying.

b.     The quality of formamide is important.  The formamide from CMS is very good in our hands.