Week 5

 During the last week of June, my mentor Ben Gregory was out of the office for a vacation. As such, I have been tasked with catching up on the backlog of PCR and gel electrophoresis necessary to identify the difference between Cx. restuans and Cx.pipens. Normally, during the week, I have performed two PCR’s per week; however, with six different trap nights coming in a week, and the preference to run smaller, 36 sample groups from each trap night, it has been challenging to keep up. 

On Monday, Izi and I ran three PCR’s. Then I had to go set up traps at the last three sites of June; Hillcrest Park, SERC, and Church of the Holy City. 

The Hillcrest Park gravid trap was set up between two trees next to a pond. The park happened to be windy, and I got dust in my eyes!

On Tuesday, we ran more PCRs and got 100% amplification when viewing the gels. We also had to make more gels for gel electrophoresis on Wednesday.


Here is the 100% amplification gel! I was so proud of the teamwork Izi and I did to get 100% amplification.

There are many different types of gels that I learned to make this week to visualize all the PCRs. The most common one made is a 2% thick agarose gel with ethidium bromide. Ethidium Bromide is a mutagenic chemical that helps to visualize the bands of DNA during gel electrophoresis. The other chemical used to do this is gel red, which was used when we made a bleaching gel as a Quality control procedure when learning how to do an RNA extraction, but I will discuss how RNA extraction works next week.  To make a 2% thick gel, 470 ml of reverse osmosis water is combined with 20 ml of 25X TAE buffer and 10 grams of agarose. This mixture is then heated for three to four one-minute intervals while stirring. Once the mixture is cooled down enough to hold the beaker in your hands, 20 µl of ethidium bromide is added, and then the gel is left to set for thirty minutes in a gel rig, but this time changes depending on the type of gel, and the size of the gel rig. Air bubbles that form during the pouring of the mixture are pushed to the walls of the gel rig and popped. If not, they can prevent PCR product from running down the gel properly, and smear the bands. 


Here is a picture of me stirring the agarose mixture between heating intervals in the microwave. The goal is to heat the mixture until it begins to bubble.

Here is the final product of 2% thick agrose gels made.

To set up a gel electrophoresis, which I did again on Friday the gel is cut to have three to four more wells than the PCR product will fill, in this case, forty. The gel is loaded with 8 µl of PCR product, including controls with a 10 µl micropipette. Next, the DNA ladder is added, skipping a well for easy visualization. 

That is all I did this week! come back next week to hear more about lab techniques and field work at the Fritz lab and UMD.

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