Template:Assemby Protocol

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