Week 5 at the Deo Lab-- Zui Dighe

This week I repeated my PCR reactions. There are two sets of reactions, the first PCR and the nested PCR. The first PCR is supposed to amplify target DNA, guided by the primers created. You must go through the full sequencing process one time to make sure that the primers created are amplifying the right products. Once the first PCR reaction shows the right bands, the nested reaction is another test to make sure that the bands are correct. With nested reactions you use nested primers which  are more specific to the regions needed.

On Monday I came in extra early to repeat my RT PCR reaction (turning rat lymph node RNA into DNA). I was the first one in the lab! It took me some time to gather all the materials on my own and I felt independent being able to work on my own. After completing the RT PCR (it takes about 5 hours) I began my first PCR reaction to test if my RT reaction was successful.

The results of my first PCR seemed to be at the right size or slightly large (successful!). Thus, I proceeded to the nested reaction on Tuesday. The nested reaction; however, shows no clear bands to the actual size of the product amplified. This is because the band witnessed is a blurred elongated one ranging from 200 to 800 base pairs. This could be amplifying what is needed but it is not completely determinable.

Thus, my PI advised me to continue the cloning with the first PCR reactions rather than the nested one.

I also realized how sensitive the RT PCR samples are to temperature. Thus, I aliquoted my samples into smaller one-use tubes as to not waste the entire stock. Hopefully, the samples will not get contaminated and I will not have to repeat the 5 hour process of turning RNA to DNA.

Here is a picture I took of one of the animals I saw on the animal floor in the CRVI building. This amphibian (the Mexican axolotl) can regenerate their limbs after an arm or leg is cut off. A neighboring lab has a grant from the army to research regenerating limbs in the field of biomedicine.