Template:Assemby Protocol

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These numbers have been based on Macherey-Nagel 8-strip miniprep of assembly vectors:

  • Set up the digestion:
 1 uL lefty plasmid(methylated)
 1 uL righty plasmid (methylated)
 1uL NEB2+ATP
 0.3 uL XhoI
 0.3 uL BglII
 0.3 uL BamHI
 6.1uL Water
  • Digest for 1 hour at 37C
  • Heat kill at 65 for 20 min
  • Add 0.3uL T4 Ligase
    • Ligate at room temperature for 30 minutes
  • Put on ice for transformation
  • Transform into pir-Righty or pir-Lefty cells (in the -80 in green or yellow tubes with red stripes along the top), rescue, plate on dual antibiotic plates
  • You MUST use pir-strains, or the plasmid will NOT be replicated.

Notes:

  • This requires the use of BglII and BamHI methylating strains!
  • 10X "NEB2+ATP" is a homemade buffer present as aliquots in 500uL tubes labeled with a red slash
  • You can make up a mastermix of the enzymes/buffer/water and distribute the plasmids into it
  • The leftmost thermocyler in 327 has a program JCA/1123 that runs the 1hr at 37 and heat kill steps of the procedure
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