Generating DNA Probes to Label the Sex Chromosomes of Anopheles Mosquitoes and Preparing the Hybridization Solution

Begin by using a commercial extraction kit to extract the genomic DNA from the pupae or from adult male Anopheles mosquitoes. Next, prepare the PCR reaction mixture. Label the 18 SR-DNA and satellite AGY 53B by performing a PCR reaction.

For the preparation of the hybridization solution, add five microliters of labeled DNA probe to a 1.5 milliliter tube. Then add five microliters of salmon sperm DNA. Precipitate the DNA probe by adding two volumes of 100%ethanol and 0.1 volume of three molar sodium acetate.

Keep at minus 20 degrees Celsius for at least 2.5 hours. Centrifuge the tube at 17, 000 G for 20 minutes at four degrees Celsius. Then remove the ethanol and air dry the pellet at room temperature in the dark for 20 minutes.

Next, prepare the hybridization buffer in a 1.5 milliliter tube. Vortex the solution for one minute and allow the dextran sulfate to dissolve at 37 degrees Celsius for 30 minutes. To obtain the hybridization solution, dissolve the DNA palette in 20 to 30 microliters of the hybridization buffer.

Vortex the solution for one minute. Perform a quick spin, and store the tubes at 37 degrees Celsius in the dark.