Template:SBB-Protocols EcoBamTransfer

From OpenWetWare
Jump to navigationJump to search

EcoRI/BamHI Transfer

1. Set up digest:

  • Set up the digest (you can do mastermixes of the water/buffer/enzymes):
 For BBb transfers:
 5 uL ddH2O
 1 uL NEB2+ATP Buffer
 0.5uL EcoRI
 0.5uL BamHI
 3 uL miniprepped plasmid
  • run temperature program on thermocycler of 1hr at 37 and then 20 min at 80

2. Add 0.5uL of predigested vector and 0.5uL of T4 DNA ligase

  • Incubate 30 min. at room temp

3. Transform into correct cell line
4. Rescue for 45min to 1hr in 37C shaker
5. Plate on correct antibiotics