Retroviral Cloning Protocol

Cloning of Retroviral Vectors (pMIG)

Digesting Plasmid

• Digest 3ug of DNA to a final volume of 60ul, for 4 hours
• Before the last hour of digestion, 1ul of CIP
• To generate blunt ends: at RT add 2ul of 10mM dNTPs, and 2ul of Klenow.
• Incubate for 20 minutes at RT
• Immediately run on a 1% low melting agarose gel

Ligation

• add 11ul of insert and 4ul of vector
• 4ul of buffer (either NEAB, or Gibco; with PEG)
• 1ul of NEB high conc. Ligase (2,000,000 u/ml)
• Incubate overnight at 16ºC.
• Add 80ul of water, 40ul of NaOAc (3M), and 300ul of EtOH
• Spin for 10 minutes at 14k; wash 1x in 70% EtOH
• Resuspend in 5ul of water, use entire volume to transform Nova-blue bugs.

Gel Ligation

• Do digest as described above
• Run fragments on a 1% low melting agarose gel=
• Cut out fragments in as small a piece as possible
• Heat gel to 70ºC for 5 minutes, vortex, transfer to 37 degree bath for at least 1 minute
• Set up ligation: add 2ul of 10X buffer, 2ul of ligase, 11ul of insert and 4ul of vector (in this order). Incubate at room temperature for 2 hours.
• Add 200ul of transformation buffer (10mM Tris pH 7.4, 10mM MgCl2, 100mM CaCl2). Heat to 70ºC for 5 minutes, transfer to ice bucket for at least 5 minutes.
• Add 20ul of NovaBlue competent bugs. Leave on ice for 10 minutes.
• Heat shock at 42ºC for 50 seconds, transfer immediately to ice bucket for 2 minutes.
• Add 1ml of LB (no amp), leave at 37 degrees for 30 minutes.
• Spin bugs and remove all sup except for 100ul; plate on LB-AMP.