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.
- 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