Week 7

 During the second week of July, Dr. Fritz's lab ran out of Ben’s squish prep buffer along with positive Culex species controls. Instead of PhD student Ben Gregory preparing more of these solutions for me, he thought it would be best to teach me how to make the buffer! The protocol to make more of these reagents requires proteinase and RNAase-free water that is communally shared in the lab. To use this water we had to pour the bottle high above an Eppendorf tube before using a graduated cylinder to avoid any contamination. Now that I know how to make squish prep buffer myself; I am better able to act independently in the lab. But there was another problem… without positive Culex restuans, and Cx. pipens controls, the PCR protocol could not identify the cryptic species! Therefore we had to do a protocol that Ben dreaded doing; DNA Extraction using the Qiagen DNeasy Blood & Tissue Kit. Just like the RNA extraction procedure I learned two weeks ago this procedure required a tedious spin column, but differed as this DNA extraction procedure was a two-day process. Overall, I am grateful to have been allowed to learn how to use this Qiagen test kit. 

To start the procedure on Wednesday, we dissected the abdomens of 10 mosquitoes from two different trap nights. 24-1 was used because it was statistically safe to assume almost all the mosquitoes would be Culex pipens, while 24-18 was used for Cx. restuans. These abdomens were put in their angle-bottom tube and labeled individually. Then, a master mix of ATL and proteinase K was added to each abdomen, and a pestle was used to grind each abdomen mechanically. The samples, along with the controls were then centrifuged and placed in a hot water bath to be retrieved on Thursday. 



Additionally on Wednesday, I sorted the gravid traps set on Monday and found a surprise! Two spiders. They didn’t seem to have eaten any of the Culex and Ades in the trap… but they were definitely planning on it! At least 5 mosquitoes got tangled in webbing.




Here is the Chain of Ades and Culex found from Waterloo Park in Elkridge, MD.



On Thursday, Ben, Izi, and I continued the Qiagen DNA extraction procedure. I realized that I needed to take more pictures of the steps of the procedure so here are some pictures illustrating the Day 2 protocol. 

Here is a picture of me loading samples into the centrifuge after the hot water bath
                                                        taken by PhD student, Ben Gregory. 



Here is Izi and I working together to add Rnase A to each sample and inverting each sample 25 times.
 


Here is a picture of me loading the centrifuge again to run the samples on low speed. PhD student Ben supervised and helped to take pictures of the process and making sure Izi and I are performing the Qiagen correctly.

After this, the samples were incubated in a warm water bath at 37 celsius for thirty minutes. While this happened, the team took a break. 

Here is Izi and Ben working to prepare the AL and ethanol master mix used in the spin column.

This spin column was done multiple times before eluting the DNA product with water. It was quite the lab protocol to do in a day!


Thats all for now, Thanks for reading!













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