Colony PCR

April 17, 2021

Catie Fleischer and Carter Beck

Introduction:

We are part of the Cancer an Immunity Stream with Dr. Land at Minnesota State University, Mankato. Our research is focused on how APOBEC3A and APOBEC3B interact with miRNAs (replicated fragments that may interfere with the expression of mRNA genes). The miRNAs that we are working with are hsa-mir-29a-3p and hsa-mir-655-5p, which have been found to be involved with a variety of different cancers.

Specific Content:

Over the past two weeks we have been working on screening our pMiniT-miRNA-29a and our pMiniT-miRNA-655 colonies using a method called colony PCR, which was done to see if the E. coli bacterial plasmid we are using picked up our miRNA inserts (amplicon) we added in a previous lab. We also started prepping some of our E. coli colonies from the PCR for sequencing.

To do the colony PCR procedure, we prepared two LB Amp plates (one for each miRNA) to grow the new E. Coli colonies on. We divided each plate into six sections to separate the six different E. coli colonies we would add. We also labeled 13 PCR tubes one through six, so we could have two sets of six tubes for each miRNA and one control tube labeled “c”. Next, we made a master mix with Taq, water, forward primer, and reverse primer and split that equally among the 13 PCR tubes. Then we used a pipette tip to add a single E. coli colony to each PCR tube except the control by swirling it in a PCR tube then making a squiggly line on the corresponding section of the LB Amp plate. Then we put the 13 PCR tubes into the thermocycler for the necessary program. While that was running, we prepared a gel to run our PCR products. Once the thermocycler was done running the program, we added the PCR products into their respective wells (image shows Catie loading the wells) and a ladder for each and ran the gel. Once the gel was done running, we imagined it, and the results were not great. Most of the PCR products did not show up at the expected band size had they picked up the miRNA inserts, so we had to re-run the PCR products. However, even though our gel didn’t turn out the best the first time we still had three PCR products that we thought we could use for the sequence preparation since the LB Amp plates we grew the PCR colonies that we used on had growth, so we started to prep those the following week.

To prepare the plasmids for sequencing, we used the PCR results to determine which plasmids we should prep for sequencing. We decided to use two PCR colonies from miRNA 655, and one PCR colony from miRNA 29a. We created a solution for the E. coli colonies we choose to grow in overnight. Then we added 2 mL of that solution to three tubes. Then we scooped up the PCR colonies we needed with a pipette tip and dispensed the pipette tips into the three tubes and left the E. coli colonies to grow overnight.

The next day we went to the lab to re-run our colony PCR products with the same procedure as earlier to get better results and see if those results would confirm the colonies we choose to prep for sequencing. We also continued on with the sequencing prep process while the gel was running, but we did not get far because our gel didn’t turn out quite right. The gel did not show any bands for miRNA 29a around the expected band size 559bp had those PCR products picked up the amplicon. However, the gel did show faint bands around the expected 609bp had the miRNA 655 PCR products picked up the amplicon for PCR products 2 (P2) and 3 (P3). The gel also shows a few faint bands around 300bp for the 655 miRNA.

After reviewing the results of the gel, we were able to go over them with Dr. Land, who agreed that the results were not the best and said we should remake our colony PCR products and run another gel. We re-did the colony PCR procedure as described above except this time we used colonies from the two plates we made while doing the colony PCR the first time because those were sectioned colonies that were all the same, and we could use the corresponding numbers since everything was labeled one through six (image shows Carter adding the E. coli colonies in the PCR tubes). Then we loaded the PCR tubes in the thermocycler to run the specified program, and once that was done we stored the PCR products so we could finish running the gel next week.

Reflection:

We definitely faced some challenges this week with our colony PCR not turning out the way we wanted, but assuming the third gel we will run ends up looking good we will move forward with prepping our E. coli colonies for sequencing. This lab was a good example of how even though you may do everything according to the procedure, stuff can still go wrong and not turn out how you would like it to. As we’re nearing the end of this semester’s research, that is definitely one lesson we have learned from this research experience.

Posted in Uncategorized | Leave a comment

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.

Posted in Bacteria & Disease | Leave a comment

Our Progress with Flavobacterium psychrophilum

April 16, 2021

Emily Schmidtbauer and Kaela Wierenga

Introduction:

In this blog post, we will further analyze our endonuclease gene and look at what our next steps have been in our research since April 1, 2021. We are still looking at the Flavobacterium psychrophilum bacteria with our endonuclease gene which catalyzes the cutting of nucleic acids.

Specific Content:

When conducting research on our endonuclease gene, little was found. We were able to find information on what an endonuclease is and its function, but little is known about how it relates to the virulence of Flavobacterium psychrophilum bacteria. The endonuclease gene is not a restriction endonuclease meaning it will randomly cut sections in a nucleic acid sequence. Because of this randomness, we hypothesized two ideas. The first idea was the bacteria could digest the DNA nucleic acids from the fish cells to smaller fragments and use these as nutrients. The second idea was the bacteria would destroy the host DNA, leaving the nucleic acid incomplete, meaning the host cell is not able to replicate the nucleic acid sequence, resulting in no cell replication and a depletion in cell count and organism growth.

Figure 1. Emily and Kaela work to transfer each of the five E. coli samplesinto a microcentrifuge tube.

After transformation of the DNA ligation product, on our E. coli plates, we had a significant amount of growth, so we were good to continue with our research. We each took one fresh LB agar plate and split the plate into ten sections. Using a sterile pipet tip, we picked up one colony on our growth plates and streaked it onto our new plate in one section. After disposing the pipet tip, we repeated the process until all our ten sections had a colony spread on it, having twenty sections total between our two plates. We then incubated our new plates for twenty-four hours at 37oC. The result was quite a bit of growth, so we picked out five sections from each plate to conduct a PCR test on.

Figure 2. The top row is our original agar plates with E coli growing after transformation. The bottom row is the ten colonies we each streaked onto a plate.

The purpose of the PCR and running a gel was to know which colonies took our ligation product. In our gel, we included a 1 kb ladder, control sample with genomic DNA as the PCR template, and our ten samples. All our samples were PCR positive and the expected DNA bands appeared on our gel, but only about half were very bright. We continued with two colonies that we think were the brightest on our gel and streaked each on a new LB agar plate for isolation of pure cultures. We incubated the plates at 37oC for roughly twenty-four hours. After we saw colony growth, we did an inoculation into a test tube with five milliliters of LB broth and five microliters ampicillin. Since our plasmid is ampicillin resistant, only the cells with our plasmid would grow. We shook our sample at 175 rpm in an incubator at 37oC  for twenty-four hours. After this was completed, we transferred 1.5 milliliters from the incubated test tubes into a microcentrifuge tube and completed the plasmid extraction process.

Conclusions/Reflections:

Little is known about the endonuclease gene on the virulence of Flavobacterium psychrophilum, but we believe our research can show if they are related. We completed the screening process of our transformed E. coli cells and found several colonies that took our correct ligation product. We continued with two colonies and extracted the plasmids. As we are soon concluding with the downstream region of our endonuclease, we are going to trouble shoot the upstream region and start over our whole process, but with new primers.

Posted in Bacteria & Disease | Leave a comment

Lab Progression

By D’Aaliyah Johnson & Matthew Trenne

April 13, 2021

Introduction:

During the past few weeks since our last blog post — February 27, 2021 — we have continued to work with our miRNA’s, Hsa-Mir-4297 and Hsa-Mir-1207 3p, in the Cancer and Immunity Stream with Dr. Land. Since then, we have encountered some setbacks, but there have been so many improvements that outweighed our hardships. The process of redoing specific labs and figuring out what went wrong has shown us how science can be unpredictable and gave us the strength to work through it as a team.

D’Aaliyah Johnson arranging PCR tubes into the thermocycler for lab twelve. (04/07/2021)

Specific Content:

In our eighth week in the lab, we ended up amplifying our MiRNA and running them in a gel to determine if our MiRNAs had indeed amplified. Our expected banding was at 300bp, and our MiRNAs did indeed show banding at that size; however, so did our controls leading us to believe that we cross-contaminated the samples at some point.

The following week we ran the same experiment as before, trying to amplify our MiRNAs following the same process, and this week our gel came out perfect. We saw banding exactly where it should have been, around 300bp, and both of our controls were negative. In the lab, we also worked on bacterial transformation; our goal was to introduce a new plasmid into the E. coli DNA that we were working with. After working through the lab, we ended up streaking bacterial plates with our E. coli samples; both of our active E. coli plates had good growth and were what we were expecting. Our control plate that did not have the plasmid also did what was expected and did not grow on the plate.

Matthew Trenne placing the electrophoresis gel into a machine to determine if there are any bands present.

The following week our goal was to isolate and amplify both our E. coli sample and finish the amplification on our MiRNA. Both procedures followed very similar protocols, and we were able to do both at the same time. After the prodigal was completed and we thought our samples were amplified, we then took the samples to the nano dropper to determine if the ratio of everything was what was expected. On both of our E. coli samples and both of our MiRNA samples, our numbers looked terrific.

The previous week we worked with an E. coli sample that was a little more forgiving; this week, we worked with a different E. coli strain that is a little harder to work with. We changed to this new E. coli because it will duplicate what we want it to faster and better. We worked on inserting our specific MiRNAs into the E. coli to create copies of our MiRNA.

That brings us to this week; we are working with our new E. coli and amplified miRNA. We are taking the E. coli that was cultured on plates last week and introducing our MiRNA, and running a gel on them to see if we can determine where banding is going to be.

Reflection:

We have learned a lot so far during lab. It was brought to our attention early on that labs will not always go as expected. We have had to encounter the long hours of hard work and dedication to get the results we wanted, and now we have begun to experience loads of success in the lab. Protocols were repeated, some more than others, but in the end, we were able to achieve fantastic data and acquire new skills that we would not have gotten elsewhere.

As of right now, we have gotten great bands on electrophoresis gels, good numbers on our nano drops, and plenty of colonies of bacteria. Within the next three weeks, we will hopefully have the appropriate results to pause for the summer. Once we come back in the fall, we will be ready to pipet miRNAs once again!

Posted in Cancer & Immunity | Leave a comment

Bacteria Growth Since Ligation

4/7/21

Abby Mueller and Marley Fuhrman

Introduction:

In our previous blog post the last procedure we did consisted of ligating our gene with the plasmid. Since then, we performed a transformation procedure and then grew the bacteria. We have seen bacteria growth, which is a good sign as we move forward. 

Specific Content:

After we finished the ligation process, we did a transformation procedure in E. coli to fuse our gene with the plasmids that we have been working with. We then used LB broth as a growth medium forour transformed E. coli, and we spread these bacteria onto agar plates. To get bacteria colonies with our gene ligated to the E. coli plasmid, the agar plates contained ampicillin which would kill any E. coli cells that did not contain the plasmid. After 24 hours of incubation, we revisited our plates and saw that a total of 8 colonies had grown on the two plates. Since we needed at least 10 colonies to do the patching process, we decided to spread the leftover of our transformed E. coli on 2 more agar plates and incubate them for a day to grow more colonies.

Figure 1. Marley places tubes into centrifuge during the transformation procedure.

The second round of plates came back with much better results than the first couple, with each having over 20 colonies. So we each picked 10 colonies from our plates and patched them onto an agar plate with numbered sections, with one colony getting patched into each section. We did this to further isolate the colonies and create a bigger surface area for them to grow so they would be easier to harvest later. We incubated the new patched plates for 24 hours and results can be seen in the attached image (Figure 2).

Figure 2. Growth of E. coli colonies that were patched onto agar plate with numbered sections.

Next, we scraped 1/4th of the cells from 5 of the best colonies on each of our plates and suspended them in water in a tube so we could boil them. The purpose of boiling them was to break the cells and release the plasmid DNA that contained our gene. We only boiled them for 7 minutes before they were cooled then put on ice. We then used the DNA plasmids that were released from the previous step as the templates and created a PCR for each different E. coli streak, plus a positive control that contained genomic DNA of Flavobacterium psycrophylum. 

Reflection:

These past few weeks have yielded positive results. We have seen a lot of bacteria growth and we expect to see positive results on the colony PCR gel we will run this week. Once we have successful plasmids for the downstream region, we will move on to cloning the upstream region of our target gene and perform the same process of E. coli plasmid extraction, DNA digestion, ligation, and transformation.

Posted in Bacteria & Disease | Leave a comment