Lab Blog Week 2

 Introduction:

This past week in lab, I worked alongside Aland and Micheal in their project with the 7KB plasmid from Aquaticus and the CMR gene from the P-RAD1 plasmid in E. coli. I got the chance to break down the process of the PCR machine and better understand what factors are involved within DNA replication. We worked with the electrophoresis process, to see what method of 7KB collection to move forward with, also giving me a chance to see step-by-step how to complete the procedure on my own. I learned the recipe for LB broth, and how to distribute them equally amongst the agar plates.

Method:

Working with Micheal and Aland, I got the opportunity to learn about their project, and learn how to perform some of the procedures involved with its experimentation. We worked with a plasmid native to aquaticus called 7KB. The aim is to add the CMR gene from E. coli onto this plasmid, giving it resistance to chloramphenicol, a powerful antibiotic.

We first had to establish whether it was more effective to extract the M23 gene from the 7KB, or just use boiled cells to access it. We completed two rounds of electrophoresis to determine this. Our first round showed results that put into question the validity of our PCR steps, since we didn’t get the results that we should have expected to see from the M23 samples. In the next round of electrophoresis, we adjusted the sample size concentration from 2μg to 10μg.

Micheal outlined the steps to creating the one percent agarose gel. We first weigh out 0.3g of agarose using the scale and put it into a flask. We then pour 30mL. of TAE into a graduated cylinder, to transfer into the initial flask up to volume. Microwave for thirty seconds and check for clarity to see if more time is needed. Before pouring, we set up the other electrophoresis materials. We take the Bio-Rad holder and place it in the two stoppers firmly against a plate, which is to be placed against the indentations along the holder. We then pour the prepared gel from the corner of the plate, after it has cooled to a comfortable tea-like temperature. After the gel is prepared, we take the pipettes and put 6μL of each sample into the well. We started with the ladder, followed by the 7KB extract and 7KB cell sample, with the M23 extraction, and M23 cell sample coming after. Once loaded, we close the lid, plug the corresponding cables to the electrophoresis machine, and start up the process. We started at 75 mA and adjusted to 80 to speed up the results. After waiting for the electricity to take effect, we unplug the machine and take out the well plate to be viewed under a fluorescent light. The results showed a stronger presence of base pairs from the M23 cell sample, letting us know to move forward with the cell sample over the extracted sample.

We then made an LB broth with ampicillin, in hopes that when adding E.coli, the ampicillin resistance from the P-RAD1 gene will be what is left, so we are able to further isolate the CMR gene from the plasmid. For LB broth, we first fill a graduated cylinder up with some water. We then weigh out 2.5g tryptone, 1.25g yeast, and 2.5g NaCl. After adding all ingredients back into the graduated cylinder, we fill the rest with DI water up to 250 mL and ensure everything is well mixed. After completing the LB media, we add 15 μL/mL of the Ampicillin and shake it up. We poured these contents into agar plates and waited for everything to cool before stacking the plates together by 4’s, and para-filming them into place. We placed our clean media in its designated spot in the right fridge.

Results:

(Figure 1. Above shown is the results of our second round of electrophoresis. To the very left we see a much more prominent reading for the M23 cell sample compared to the M23 extraction sample.)

Discussion:

It was very important that we got the 7KB data to show up correctly during electrophoresis, since that would show that our primers were being built correctly. When the first run didn’t show the results we expected, we investigated what possible step taken was faulty. We ensured the master mix gave enough nucleotides to build the primers as it should, and double-checked temperatures during the annealing stage. I think one way we could have ensured the most amplification of primer building during the annealing stage would have been to run multiple samples on the same gel through with different temperatures, and see when it lights up, to determine the most effective annealing temperature. We thought when nano-dropping the samples, something could have also gone wrong with the reading it gave, and its accuracy, so we decided to up the concentration for safe measure. Next week, I am going to try to get a better understanding of the goal of this project, and anything else I’m a little unsure about. In lab, I also plan to work with the mentor students to have them teach the function behind a few of the machines found in the class. I hope that next week, I get the opportunity to see the LB media & Ampicillin combination in action with the E coli,, as I am curious to how it will isolate the P-RAD1 Plasmid