Lentiviral Cloning Protocol
Lentiviral shRNA Cloning Protocol (pLL3.7, 3.72, 3.77, pSICO, pSICOR)
Oligo Preparation
- 1ul Sense Oligo
- 1ul anti-sense oligo
- 48ul Annealing Buffer (100mM K-acetate, 30mM HEPES-KOH (7.4), 2mM Mg-acetate)
- Either use PCR machine to heat to 95 C for 10', and then slowly cool to RT, or heat in heatblock for 10' and then remove from heating unit and allow to cool to RT.
Vector Preparation
- Digest 5-10ug of vector DNA with Xho1 and Hpa1 for 3 hr at 37 C
- Add 1ul per rxn of CIP, incubate at 37 C for 30'
- Quickly run rxn after CIP incubation on 1% agarose gel at 180V for ~45 min
- Purify cut vector band via Qiaex Kits
Ligation
- 11ul of oligo rxn
- 4ul of purified and cut vector
- 2ul of T4 DNA Ligase
- 2ul of T4 DNA Ligase Buffer
- Incubate overnight at 4 C
Transformation Preparation
- Add to ligation rxn tube 80ul ddH20, 40uL 3M NaOAc, 300ul 100% EtOH
- Spin 10' at 14k
- Wash with 70% EtOH
- Spin 10' at 14k
- Remove EtOH and allow tube to dry
- Resuspend pellet in 5ul of H20
- Transform resuspended ligation rxn in 20ul of Nova Blue Competant bugs
