BIOTIN LABELING AND HYBRIDIZATION OF PROBES TO CHROMOSOMES
Suggested chemicals: Nick Translation Kit,Enzo DiagnosticsCat. No. 42803.
Biotin labeling of plasmid or genomic 1xbet sports betting
- Remove 1xbet sports betting from -70°C freezer, remove the cover slips with a razor blade, and dry the 1xbet sports betting in an ethanol series (70%, 95%, 100% ethanol; 5 min each at room temperature). 1xbet sports betting should be dried for several hours before ISH.
- Prepare the following hybridization mixture. 10 µL of the hybridization mix is used for one slide and covered with an '18 x 18' cover slip. Care should be taken that the sloution is well mixed, because the 50 % dextran sulfate solution is very sticky.
| deionized formamide | 10 µL |
| 20 X SSC | 2 µg |
| sheared salmon sperm 1xbet sports betting (10 mg/ml) | 2 µL |
| probe 1xbet sports betting | 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 |
- For genomic ISH, prepare the hybridization mixture as below. The amount of the sheared blocking 1xbet sports betting is the most critical part of this procedure. The blocking 1xbet sports betting should be in excess of 100 to300 times more than the probe 1xbet sports betting .
| deionized formamide | 10 µL |
| 20 X SSC | 2 µg |
| sheared salmon sperm 1xbet sports betting (10 mg/ml) | 2 µL |
| probe 1xbet sports betting | 1 µL |
| blocking 1xbet sports betting | 1µL |
| 50 % dextran sulfate | 4 µL |
| Total reaction volume (use H2O to adjust the total volume to 20 µL) | 20 µL |
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 1xbet sports betting (by using a slide holder that can handle 10–20 1xbet sports betting ) 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 1xbet sports betting into an ethanol series (70%, 95%, and 100%, 5 min each at 20°C), and air dry the 1xbet sports betting .
- Add 10 µL of the hybridization mixture to each slide and cover with an '18 x 18' mm cover slip. Place the 1xbet sports betting 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 1xbet sports betting ).
- Incubate the 1xbet sports betting in the moist chamber at 37°C for a minimum of 6 h or overnight.
