Contributors: Esther, Chloe, Alexander, Emma, Tank
GC-rich optimised faithful PCR
For inserts where the standard Q5 protocol failed, a GC-rich-optimised variant was tried (5X Q5 buffer, 200 µM dNTPs, 0.5 µM each primer, <1000 ng template, 5% Mg²⁺, 1X High-GC Enhancer, 5–10% DMSO, Q5 HiFi polymerase). Program: 98 °C 30s → 35 cycles of [98 °C 10s, 72 °C 2.5 min combined anneal/extend] → 72 °C 2 min final extension → 4 °C hold. Redid the faithful PCR of the insert with this protocol.
M9 plate prep
Two 500 mL Duran bottles prepared in parallel (one to be autoclaved, one not): 62.5 mL 4X M9 + 157.2 mL water + 7.5 g agarose, with 0.25 g yeast extract added to the autoclaved bottle only.
The non-autoclaved bottle’s agar came out visibly more viscous, with an observable agar layer — since yeast extract shouldn’t increase viscosity, this points to contamination (excess agarose relative to what was measured). This may explain the brown-tinted plates seen in earlier M9 work (possible glucose/Maillard-type contamination).