Continuing Plasmid Amplification and Injection Mixes

For the past 2-3 weeks, I have been altering PCR conditions to identify which conditions provide the cleanest and most desired DNA. This involves doing several temperature gradient PCRs, as well as gradient PCRs with varying concentrations of DMSO.

This few weeks of doing PCR, although being repetitive, I have learnt is an important step to this research. Ensuring that PCR conditions are optimised allows the correct insert, and the correct amount of insert to be injected into our worms for screening.

Despite having identified what we had thought were the optimal conditions for PCR amplification of the insert, when running these conditions individually, we have found that these conditions weren't actually optimal. These supposed optimal conditions resulted in several bands, leading to further optimisation of these PCR conditions. We finally opted for PCR conditions that although did produce more than one band on a gel, however produced the cleanest bands we could achieve. These conditions wer, 67.4C, 5% DMSO for SAS-6 and 66.4C, 3% DMSO for SPD-2. This process was particularly tough as most of it was repeating the same process everyday until it worked. However, as I stated before, this process in particular is important, especially in biology as not everything will work and I should keep trying again until I get the right outcome.

As of today, the PCR amplification conditions have not been fully optimised for SAS-4, however, has been optimised for SAS-6 and SPD-2. This allows me to progress onto the sequential steps of this project for these two genes. This includes the preparation of the injection mix that will be used to inject the insert into the worms. This injection mix was prepared by purifying the DNA using QIAGEN PCR purification kits and AMPURE XP beads.

Purification of PCR produce using AMPURE XP beads

This process was interesting as the beads bind to the DNA, allowing me to clean off and remove any buffer/PCR mix left with the DNA. As one of my supervisors describes, these beads are "magic" and effectively purifies the DNA so that we are left with (hopefully) just DNA suspended in sterile water. The purified DNA was then added to a CRISPR mix and roller plasmid (to provide a clear phenotype to see if the injection had gone into the worm).

Once the injection mix was made, it was kept at 4C until it was time for the injection.