Figure 1. Pipetting water into the dried-formed plasmid fragments.

Unexpected Circumstances

All ordered materials (gene fragments from Twist, primers from IDT) arrived on the Thursday before Tuesday’s presentation, leading to a tight timeframe.

I’ve previously researched and planned my Lab Protocal, yet several things did not happen as planned:

• The gene was delivered in multiple fragments, requiring Gibson Assembly, contrary to my initial expectation of receiving a fully assembled construct.
• The TX-TL cell-free kit was unavailable, requiring a shift to in vivo testing using E. coli.

As a result, I revised my plan:

• Shifted from cell-free and textile testing to in vivo testing using E. coli
• Focused lab time on assembling the full genetic circuit and performing diagnostic validation

Fig.2 The plasmid fragment ordered from Twist Bioscience (green cap) and the their primer pairs (yellow cap)

Day 1 - PCR

Ren offered me great help in walking me through the processes, and explaining to me on how to calculate the measurements and PCR temperature from thermal fisher’s online calculator.

Fig.3 Ren’s note on PCR and backbone digest

1. Preparing the mixtures for PCR

First, rehydrate the Twist lyophilized DNA fragments in nuclease-free water to the final working concentration (10 ng/µL):