Return of miRNA-4314 & miRNA-4786-3P for Fall of 2021

October 19, 2021

Aidan Forberg, Lauren Buysse, Eli Landas

Introduction:

Last semester, we successfully inserted our miRNA hsa-mir-4314 into the pMiniT plasmid. For our other miRNA, hsa-mir-4786-3P, we did not receive positive sequencing back, which has us behind where we would like to be. This is a summary of how much progress we have made so far this semester. We are excited to be continuing our research this semester with fewer COVID-19 restrictions.

Figure 1 – Our group working on the UV lightbox to cut our gel for gel purification.

Specific Content:

We began this semester with a restriction digest and phosphatase treatment, with the goal of inserting our miRNA into an expression vector, pcDNA. A restriction digest allows our miRNAs to connect with the plasmids and become a part of the DNA. The phosphatase treatment keeps our DNA from rebinding to itself. In our lab waiting time, we have been trying to get our miRNA-4786 to the same spot in our process as miRNA-4314. This has involved multiple colony PCR’s, purification, and preparations for sequencing again. We still have not been successful in receiving a sequence back for miRNA 4786.

After our first restriction digest, we did not see the bands in our gel that we were hoping for. To figure out what wasn’t working, we did another restriction digest, without either of our miRNAs, in order to see if the enzymes we were using (EcoRI/BamHI) were doing their part, or if it was a manual error. We ended up with positive bands everywhere we wanted to, so we knew the enzymes were not at fault.

Figure 2 – Gel results from our protocol determining if our restriction enzymes were working.

Since we knew the enzymes were working, we ran another restriction digest using EcoRI, BamHI, and EcoRI+BamHI to determine what went wrong in the original restriction digest. to see if it was our miRNA that wasn’t working or if there was a problem with the carrying out of the protocol.

We ended up being able to see a good band in our gel for our pcDNA. We then used ultraviolet light, as well as a scalpel, to remove the band from the gel. Since then, it has been in our freezer box, awaiting further steps.

Conclusions/Reflections:

Our next steps for the miRNA 4786 are to run a PCR like our original one that worked from May 1st of last semester. The protocol will be the same for miRNA 4786 using an annealing temperature of 68℃.

A miniprep for miRNA-4314 will be done with the intent to get 1ug or more of the DNA. For this to be possible we will need a concentration of 25ng/µl but would like it to be over 50ng/µl from the nanodrop as this will be multiplied by 40µl which is the amount we will add. A larger amount of input DNA should give us less room for error in getting the expected bands on the agarose gel.

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