Q-PCR 2/19/24 - 2/23/24 Blog #5

 Introduction

During this week we learned about the fluorescent microscope and the dyes that are used for it. On Friday we prepped and did a q-pcr for our E. Coli, along with starting sugar fermentation biochemical tests by inoculating some tubes. 

Method


The biochemical test that was performed was to check if D. Sonorensis can ferment certain sugars and in this case, it was Sucrose, fructose, and glucose. The broth is a reddish color. If the sugars are fermented, acids will be produced and will change the pH below 7 of the broth therefore changing the color to yellow. If the sugars are not fermented the bacteria will consume proteins and produce alkaline change the base above 7 changing the media a dark red. On Thursday 6 tubes were inoculated while three were left without inoculation as a negative control. Three of the tubes were inoculated with staph as a positive control. The last three tubes were inoculated with  D. sonorensis. 

 Steps of inoculation plate-to-broth

-prep your area (clean and get your materials) and turn on the Bunsen burner. 

-sterilize the loop by putting it into the fire 

-let it cool then get some of the sample bacteria from your plate 

-then without putting the loop down take the cap off of the tube pass the opening of the tube through the fire to prevent contamination 

-then place your loop in the tube and swirl it into the brothers without touching the walls 

-take out the loop sterilize the opening again and put the cap back on but don't completely twist it shut as to let in the air 

-after sterilizing the loop for the final time label the tubes and clean the area again


We then did a q-PCR for our E. Coli. Q-PCR (or quantitative PCR) is a process used to detect/quantify specific gene sequences.


process for Q-PCR

-first plan what will go into your tubes

   •we made a master mix with material that each well was going to get 

-before pipetting everything out into your well plan out what will go into each well

-the in the fume hood pipette all the materials into its specific planned-planned wells 

-then cover the plate and place it into the spinner

-then place the plate in the q-PCR machine 


Result

The results for the Q-PCR were looking good while the q-pcr machine processed the sample we could see that the gene was being expressed properly 

Discussion 

While doing the q-PCR everything had to be precise. The process is meticulous, and any slight mistake couldn't ruin your process. The purpose of the master mix is to try to decrease the margin of error. Because we're working with such a small amount of liquid, and the pipetting necessary there is a lot of room for errors. I was assisting Alex by checking off what she put into each well as she pipetted. 

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