I am so sad that today is my last day in the lab. Jessie first started the day by informing me on the results of the experiments from yesterday. Unfortunately, because their were no colonies on the plate it was concluded that the results of the experiment were mostly inconclusive. The source of error Jessie believes may have caused their to be no colonies could have been to the fact that the antibiotics in the agar plates were not strong enough. As a result, for most of the morning Jessie and I created new agar plates for the bacteria colonies to grow on. After we finished creating the new plates, Jessie started the procedure of re-plating the bacteria onto the plates. Hopefully the experiment will be successful next time! Once this work was done, I finished my day by looking at my design of the NC State logo with cyanobacteria. The progress was so amazing to see. In almost 1 week there were colonies of bacteria growing around the logo I drew on the plate!
Have you ever wanted to go on a tour of NC States “state” of the art laboratory? Well you’re in luck today, for Jessie showed me all the ins and outs of every laboratory equipment in her lab! The first laboratory equipment she showed me was a machine that separated proteins by their size. According to Jessie, this piece of equipment is probably the most expensive, warranted for how impressive and complex of a machine it looks.
This machine separates proteins by their size.
The second piece of equipment was a gigantic centrifuge, which is a devise used for separating particles from a solution according to their size, shape, density, and viscosity.
Centrifuge
Most of the rest of the machines in the laboratory were used for analyzing various types of DNA in relation to the output result a given scientist desired.
After touring the chemistry laboratory, Jessie decided to start preparing samples of tolyporphin in solutions so that we could extract the DNA later with liquid nitrogen. To accomplish this result, it is crucial to crush the tolyporphin into very thin fragments to analyze. A mortar and pestle was used to crush the tolyporhphin until its green hue turned to more of a whitish color – liquid nitrogen was poured periodically to keep the substance cold and in a suitable environment for the experiment.
This step was perhaps the most intensive as it took around 30 to 45 minutes to crush the tolyporhphin to this state. Once this step was completed, the DNA fragments were extracted with phenl, a mildly toxic substance for humans, and the separated DNA fragments were extracted. To achieve accuracy and preciseness, this step was repeated about three times. Finally, to conclude the day, the separated DNA fragments were injected into a sample of bacteria to test and the results of the experiment will be analyzed tomorrow!
Unfortunately, because of a problem with the ligation and the fact that Jessi was teaching in the morning again, she advised me to take the day off today. Can’t wait to get back in the lab tomorrow!
Today was another exciting day in the lab! Because the PCR reaction and the UV test was positive, Jessie was able to continue on with the ligation reaction. In science terms a ligation refers to the joining of two DNA fragments through the formation of a phosphodiester bond. The ligase is an enzyme that catalyzes this reaction. In this experiment the two DNA fragments were the digested vector and the Digested DNA insert – they were catalyzed and joined together by the T4 ligase. After a couple of calculations, which I will list below, it was found that the master mix for the DNA vector that would contain this reaction needed 2 microliters of the buffer, 1 microliter of the ligase, 2.4 microliters of DNA, and 13.6 microliters of water. On the other hand the master mix for the digested DNA insert need about 1.6 microliters of the DNA, 2 microliter of buffer, 1 microliters of ligase, and 14.4 microliters of water. Each piece of DNA needed to be placed in a buffer in order to ensure they do not die out and are sustained in proper conditions. After each of the master mixes were created and the ligation reaction was initiated, Jessi decided to show me another crucial part of a scientists life: cleaning! Because Jessi had not cleaned her glassware for a couple of days now, she decided that it was about due time to do so today. The procedure for cleaning glassware in the lab is similar to how we clean our equipment at school. First, like most would do at home, she poured a soap mixture into each dirty beaker, then she poured acetone, and finally rinsed each glass with water.
Progress on bacteria culture!
After all the glasses were washed, it was time to prepare some base solutions. These types of solutions are used every day in the lab and it is important to keep a constant supply. The optimal pH for the base solution was about 7.5. To gain the right pH, small concentrations of HCL and NaOH were poured to achieve the desired pH. To achieve this result, Jessie used a special pH meter donated by Dr. Lindsey’s previous boss. Overall, combined with the ligation and a new found appreciation for cleaning in the laboratory, this day was filled with so much fun and action!
Have you ever wondered how to make a gel? Fortunately, I have a magical procedure for you. Ah, yes a procedure that may not make your hair slick again, but can make your DNA look slick in the chemistry lab!
This morning, I walked through the looming doors of Dabney hall to greet my first objective: create a gel solution. For Jessie,making this gel solution was important so that she could test the presence of Toly 1 - psynb inside the solution of DNA she had created earlier. To create the gel solution we used 30 mL of a special buffer and agarose. Agarose is a substance made from agar and is used specially in gels for electrophoresis. The use of Agarose gel electrophoresis is the most effective way for Jessi to separate the DNA fragments she wanted. Moreover, before we started the procedure, Jessi allowed me to create a master mix of the restrictive enzymes and buffer. The restriction enzymes were used to digest the DNA molecule and along with the agarose gel help analyze the fragments of DNA for Jessie. To separate DNA using gel electrophoresis, Jessi loaded the DNA into pre-cast wells into the gel and applied a current. Because the phosphate backbone of the DNA molecule is negatively charged, when the current was applied the DNA fragments moved to the positive charged side of the field, which was the agarose gel and buffer prepared before. Moreover, since agarose polymers are known to create a matrix of small pores, the smaller DNA molecules, were able to navigate through the mesh faster than the larger molecules. Thus, through this process, Jessi was able to separate the different DNA molecules she wanted by their size.
Procedure and calculations for creating gel and master mix
Whew that was a lot of science! Fortunately, there was lots of downtime between experiments for Jessi to share some fun facts, tips, and tricks for handling life in a chemistry lab! Below is the list I have compiled for today.
Did you know that chemists face the same struggle as teenagers at home? They also have to wash and clean! However instead of your parents watching over you, Jessi recollected that "her boss often enjoys to stroll around the chemistry lab and ensure every piece of lab equipment is spick and span". But perhaps, your boss hasn't checked your workplace for two weeks - do not worry for NC STATE's own safety department will randomly check to ensure you are in line!
Have you ever waited 30 minutes or perhaps even an hour for your salt to dissolve in your solution? Don't worry Jessi has an innovative solution that will cut that time to precisely one minute and 30 seconds: stick your solution in the microwave!
After lunch and the quick tip session, the electrophoresis was finally completed. Jessi showed me the finished product under a UV light. Although, I currently do not completely understand the method by which the UV light was used to analyze the DNA, Jessi concluded that that the DNA tested positive and that we will do a ligation reaction tomorrow!
Analysis of DNA under ultraviolet light
We may have not created a gel for your hair today, but we did create a gel that could be at the center of some revolutionary research - stay tuned for more!
Unfortunately, Jessi, the graduate student I am working with, was teaching a class today in the morning and grading papers in the afternoon. As a result, I was unable to come to the lab today. Can’t wait to step in the lab tomorrow!
Today was a very exciting day in the lab! I started my day at around 9:30 with Jessi, the graduate student for Dr. Lindsey at NC State. We first began by learning how to use various micropipeters and proper lab procedures when dealing with bacteria. Besides wearing safety glasses and a lab coat, one of the most important safety procedures when dealing with bacteria is making sure each flask that will be reused is exposed to a flame- this is to ensure no external contamination. After learning these procedures, Jessi informed me about the project she and Dr. Lindsey have been working on: extracting Tolyporphin from cynobacteria. This project is of crucial importance because currently scientists are unaware of the various functions inside the DNA of Tolyporphin and its origins inside the cyanobacteria. To truly understand this concept, Jessi taught me how to read a cell map and design a genetic sequence that can be inserted into a bacteria. Moreover, one of the preliminary experiments today was preparing solutions of old Tolyporphin bacteria cultures and placing them into new base antibiotic solutions. This experiment was an application of the micropipeting skills Jessi taught me earlier, with a focus on precision and minimizing error. For Jessi, this procedure is important so that the bacteria in the flasks are able to continue for longer periods of time and not die out.
At around 12:00, Jessi, her friend, a post-doc at duke, and I went to go eat lunch at a nearby restaurant- the food was amazing! I remember Jessi recounting to me how most days graduate students in her lab would grab lunch here and stop at the nearby coffee shop next door.
I honestly can not thank Jessi and Dr. Lindsey enough for this amazing experience. It is truly fascinating to see all the hustle and bustle of scientists throughout the university, in the labs, and across the offices - ground breaking research really is at every corner.
