User:David Johnston Monje/Notebook/Maize Endophyte Biofertilizers/2009/05/10

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The Daily Grind

  • Strategy for alSD cloning into pSDK-GFPuv. PCR alSD from glycerol stock plasmids. Cut PCR product partially with NdeI (as it has one NdeI in the middle). Gel extract the full length fragments, and ligate to NdeI cut and dephosphorylated pDSK-GFPuv.
  • Strategy for ACCD cloning : reorder primers for ACC Deaminase to introduce NdeI site on the start codon and SbfI (a compatible end with PstI) after the stop codon and an artificial termination sequence. Scratch that: already have primer for NdeI on start, and RBS plus NdeI after stop codon, so use these in PCR and clone into pDSK-GFPuv.
  • Did Phusion PCR of glycerol stock cloned genes, and will do NdeI digest on both (partial for alSD) and clone directly into pDSK-GFPuv or pSB1A2 with PSBA::GFPuv