What challenges have you had as a scientist? How have you been able to overcome these challenges?

Yesterday was a normal day. Woke up tired and grateful. Did all routines and set up for the lab. PCR these days has been proving difficult. Bacterial community analysis with ampicillin and gentamycin seems so hard to achieve with PCR not working for more than 2 months (I left it after trying twice and was doing something else).

At last, a colleague came up with a solution but with the conventional PCR protocol for 16S rRNA analysis. This protocol is different from that developed for 16S rRNA amplicon sequence analysis. There seems to be a glimpse of hope because the PCR worked, and we could see clear DNA bands on our agarose gel.

But we need to test if it works for amplicon sequencing so amplified DNA got purified (using a DNA purification kit) but DNA concentration was too low when measured with the Qubit and high when measured with the Nanodrop. Another dilemma there. Okay, let’s run the purified DNA on an agarose gel to see if we can have clear bands. Here is what we got.

Can this work for the amplicon sequencing or not? I am not sure. What can I do to improve the concentrations of m DNA for sequencing? Maybe I need to run more volumes as I initially had 25 µL or maybe I can do 3 parallels for each sample.

So back to my questions. What challenges have you had as a scientist? How have you been able to overcome these challenges?