April 17, 2021
Catie Fleischer and Carter Beck
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.
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.
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.