After purification blocked progress the previous week, our priority was to determine why DNA recovery was inconsistent and whether the workflow could be made reliable enough for assembly.
We compared digested and undigested PCR products, varied elution temperature and incubation time, and examined gel extraction alongside PCR purification. The results showed that DpnI digestion was not responsible for the contamination. A longer, warmer elution step produced more promising DNA yields, although the first apparent success could not immediately be reproduced. Further tests suggested that PCR purification gave a better balance of yield and purity than gel extraction for some samples, while gel extraction remained useful when an unwanted band had to be physically removed.
These experiments finally produced material good enough to move forward. The team completed a Gibson assembly using the linearised pBAD backbone and beta-lactamase insert, transformed the product into bacteria and began isolating colonies. This was an important shift: the project had moved beyond preparing separate DNA fragments and into constructing the plasmid needed for selection.
The error-prone PCR workflow also became more defined. By repeating the manganese and magnesium optimisation gel, we identified conditions that produced clearer amplification. Later rounds still showed mispriming, so we compared gel-extracted and PCR-purified templates and began developing tools to process sequencing data. These steps reflect an increasingly iterative approach: each round is not only intended to generate diversity, but also to reveal which conditions preserve enough usable material for the next round.
Progress continued on the tardigrade-inspired branch. We prepared induced cells and ran a first desiccation trial using saline. When diluted samples failed to produce colonies, we reconsidered the recovery conditions, including elution volume, vortexing and plating. Rather than treating the dry run as a definitive test of protection, we used it to identify practical weaknesses in the assay itself.
A power outage ended wet-lab work early on the final day, but it also provided time to review the cloning timeline, mispriming problem and stress-assay design. By the end of the week, the purification bottleneck had not disappeared entirely, but it was no longer preventing all downstream progress. We had reached assembly, generated new sequencing data and developed clearer questions for both experimental branches.