Mosquito Embryo Microinjection: A Technique to Deliver Exogenous DNA into Embryo Yolk of Anopheles Gambiae to Generate Germline Mutations

Use a micro-loader tip to fill the needles with two microliters of DNA mixture. Insert the needle into the needle-holder, and connect the automated pressure pump tubing. Align the needle so that it makes an angle of 15 degrees with the slide's plane.

Open the needle tip by carefully touching the first egg. Then, insert the needle’s tip to about 10 micrometers in the posterior pole. A successful injection will lead to a small movement of the cytoplasm within the egg.

Use the microscope co-axial stage controls to move to the next egg to continue injection. To ensure that the needle tip remains open and has not been clogged, press the 'Inject' button before entering another embryo, and visualize the small droplet at the opening of the needle. If the needle gets clogged, press the 'Clear' button to clear the needle, and repeat the droplet visualization test.

Adjust the pressure as needed, if the needle tip opening gets slightly bigger to ensure that the droplet size stays small. Inject about 40 to 50 eggs with one needle. After injections are complete, rinse the eggs off into a glass container lined with filter paper and filled with 50 milliliters of deionized water.