Biotin labeling and hybridization of probe to chromosomes

 

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Suggested chemicals: Nick Translation Kit, Enzo Diagnostics Cat. No. 42803.

Biotin labeling of plasmid or genomic DNA.

Remove slides from -70 C freezer, remove the cover slips with a razor blade, and dry the slides in an ethanol series (70 %, 95 %, 100 % ethanol; 5 min each at room temperature). Slides should be dried for several hours before ISH.

Prepare the following hybridization mixture:

deionized formamide	10 µl
20 X SSC	2 µg
sheared salmon sperm DNA (10 mg/ml)	2 µl
probe DNA	0.5-2 µl
Deionized or distilled water	X µl
50 % dextran sulfate	4 µl
Total reaction volume (use H2O to adjust the total volume to 20 µl)	20 µl

10 µl of the hybridization mix is used for one slide and covered with a '18 x 18' cover slip. Care should be taken that the sloution is well mixed, because the 50 % dextran sulfate solution is very sticky.

For genomic ISH, prepare the hybridization mixture as below:

deionized formamide	10 µl
20 X SSC	2 µg
sheared salmon sperm DNA (10 mg/ml)	2 µl
probe DNA	1 µl
blocking DNA	1 µl
50 % dextran sulfate	4 µl
Total reaction volume	20 µl

The amount of the sheared blocking DNA is the most critical part of this procedure. The blocking DNA should be in excess of 100 to300 times more than the probe DNA.

Hybridization of probe to chromosomes.

Denature the mixture by placing at 75 C for 5 min, immediately chilling on ice for 5 min, and spining down the solution.

Place the slides (by using a slide holder that can handle 10-20 slides) in 70 % formamide in 2 X SSC (140 ml formamide + 20 ml 20 X SSC + 40 ml H2O, this solution can be used several times; the quality of the formamide is important, i. e., formamide from CMS is of constant good quality) at 70 C for 2 min.

Immediately transfer the slides into an ethanol series (70 %, 95 %, and 100 %, 5 min each at 20 C), and air dry the slides.

Add 10 µl of the hybridization mixture to each slide and cover with an '18 x 18' mm cover slip. Place the slides in a moist chamber (any kind of box can be used with 2 layers of wet, 3 mm Whatman paper on the bottom; use plastic bars to hold the slides).

Incubate the slides in the moist chamber at 37 C for a minimum of 6 h or overnight.