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Refaeli Lab Lentiviral Cloning Protocol - National Jewish Health

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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

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