Week 3


During the third week, Izi and I learned about how to prepare a “squish prep” otherwise known as a DNA extraction. This step was fun to learn because it involves more technical squish prep where we must dissect the mosquitoes by separating the thorax from their abdomen. 


Picture of dissected Culex mosquito. Abdomen is used for squish prep and kept in a strip tube while the thorax is kept in a separate tube as the individual.


To prepare for this first part of DNA extraction, we set up an ice bucket to keep the thoraxes, and four strip tubes to keep the abdomen. The tubes for the thoraxes are much bigger and are labeled with the mosquito's names, which include the year, their trap night, and the number of the individual. We organize the individual abdomens using a drawn diagram. Shown below.


This is an example of the notebook setup and the microscope used to dissect the mosquitos. the beaker contains a 10% bleach solution for disinfecting the Petri dish and forceps.

Generally, it is best practice to label all thirty of the tubes that will contain thoraxes before dissection. To dissect the mosquito a microscope is used to see the mosquito's tiny anatomy, then two forceps are used. One forceps is used to hold the thorax and the other is used to pull the abdomen off of the mosquito. Between each mosquito, the forceps are dipped in a 10% bleach solution to prevent contamination of DNA. Generally, up to five mosquitoes are kept on a petri dish at a time before wiping down the petri dish again to prevent cross-contamination. 


This is an example of the tubes used to keep each individual mosquito's thorax.

The abdomen's DNA is extracted to be used as the DNA in the PCR, but I’ll explain how that is done next week. The thoraxes are used to compare the DNA from the abdomen to cross-check the species identification of each mosquito. 


For the next step of mosquito DNA extraction, 50 µl of squish prep buffer is added for each strip tube using a micropipette. To ensure the enzyme in the squish prep buffer can extract DNA, the abdomen of each mosquito must be crushed, this is done using the micropipette as a pestle in an up-and-down motion. It is quite a grip-strength workout! 


Here is a picture of me adding the squish prep buffer to each strip tube with the abdomens, and the buffer is kept in ice and vortexed during use. 


Finally, the squish prep is placed in a thermal cycler for 20 minutes to allow the squish prep buffer to extract DNA. 



Here is a picture of the thermal cyclers used for various laboratory techniques. In addition to being used for DNA extraction, it also is used for PCR.


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