Protocols: Retroviral Cloning
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.