Last Blog of the Semester

Apr 23, 2021

By: Reika Hallett & Zach Leaf

Introduction

Since our previous blog posted on April 3, 2021, we have continued working with our downstream sample of our gene. The last step talked about in the previous blog was ligation which combined our plasmid and insert into one sample: our ligate.

Specific Content

After finishing the ligation process, we then did a transformation. Transformation is the process of putting our ligate into competent E. coli cells. The reason we have to do this procedure is because it will help prepare us for the upcoming steps of screening and then a colony PCR would be performed. After transformation, we had to prepare LB ampicillin plates. To do this, we used LB powder and added water. Afterwards, we autoclaved it at 121°C for 20 min. After the autoclave, we cooled it off so we could then add the ampicillin. Finally, we poured this sample equally into various petri dishes that we could use to grow our transformed sample. The liquid we poured into these plates is called LB agar; it’s food source for our bacteria. We spread out the transformed sample onto 2 plates equally then incubated them for 24 hours in hopes that various colonies would grow. After coming back to our plates, we saw that many colonies grew so we were able to move onto the next steps. For our next steps, we made a grid on 2 different plates with 10 separate colonies on each. We then took 10 different colonies from each plate and streaked them into the 10 different grid spots on the 2 new plates which were incubated for 24 hours. Next, we scraped approximately 1/4th of the 5 different streaks from each plate to create a PCR. The PCR would tell us which colonies contained our plasmid. We would then be able to tell if they worked because the sample would show up a DNA band of around 2kb on the gel image after agarose gel electrophoresis.

Figure 1. Gel image of Colony PCR.

After imaging our gel (Figure 1), we saw that only 3R and 5R ended up working. Since these are the only 2 that contained our desired plasmid, we streaked the rest of the sample from the gridded plate and streaked it out on a new plate. On this new plate, we streaked it out to dilute the number of colonies because we only wanted one colony from this streak. After letting the bacteria grow more on this new plate, we took one bacteria colony and inoculated it LB broth. We then did plasmid preparation from the broth culture and used the Nanodrop to determine what the concentration and purity was. The next and final step we did was digestion. Digestion was done to separate the insert and plasmid that we had previously combined into our product. We digested both the 3R and 5R samples. To do this we added our 3R or 5R sample, buffer, H2O, and our 2 enzymes (BamH1 & Sal1) into one tube. Then we incubated them both for 2 hours. After incubation, we ran one last gel to see if we could see both our plasmid and insert on the image. If both the plasmid and insert turned up on the image, that would be how we would know that our product worked. We found only 5R worked as it had both the insert and plasmid showing on the gel (Figure 2). This new plasmid was named as pZR01.

Figure 2. Digestion Results.

Reflection

Now that we are finished with our downstream portion of research since both insert and plasmid showed up on the gel image after the final digestion, we can now move onto working on the upstream region of our gene. We will be redoing the same exact procedures to our upstream sample that we did for the downstream. Finally, we will be combining the upstream and downstream samples on one plasmid in the end.

This whole process has been an amazing journey so far and we can’t believe that we are already half way done. Through all the high and all the lows everything that we have done as been so fun and we cannot wait to be back in the fall to continue the second half of our research.

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