EcoRV restriction enzyme

Protocol tips

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	The amplified fragment was first cloned into a pMD-18T vector (Takara Bio Inc.) with fusion green fluorescent protein expression and then subcloned (BamHI + EcoRV; Takara Bio Inc.) into a pWPXL lentivirus vector.

Protocol tips
The amplified fragment was first cloned into a pMD-18T vector (Takara Bio Inc.) with fusion green fluorescent protein expression and then subcloned (BamHI + EcoRV; Takara Bio Inc.) into a pWPXL lentivirus vector.

Publication protocol

Pri-miR-299 sequences were amplified and cloned into the pWPXL lentiviral vector (a gift from Professor Didier Trono, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland). Briefly, a ~700 bp fragment carrying pri-miR-299 was amplified from human genomic DNA by the Phusion® High-Fidelity DNA Polymerase enzyme (New England Biolabs, Inc., Ipswich, MA, USA) using the following PCR primers: Forward 5′-CGGGATCCATGACGTGGTTGACTACGC-3′ and reverse 5′-GCGTCGACTCTTCAATTACTCCAGAGG-3′ (Invitrogen; Thermo Fisher Scientific, Inc.). The amplified fragment was first cloned into a pMD-18T vector (Takara Bio Inc.) with fusion green fluorescent protein expression and then subcloned (BamHI + EcoRV; Takara Bio Inc.) into a pWPXL lentivirus vector. The pWPXL vectors were transfected into HEK-293T cells with the packaging plasmid psPAX2 and the VSV-G envelope plasmid pMD2.G (obtained from Dr Didier Trono) using Lipofectamine® 2000 reagent (Invitrogen™; Thermo Fisher Scientific, Inc.). Cell supernatants were collected at 48 h post-transfection and passed through a 0.22-mm filter. The titer of purified virus was 2.0×108 IU/ml. In total, 1×105 NB4 and HL-60 cells were infected with 1×106 recombinant lentivirus-transducing units plus 6 µg/ml polybrene (Sigma-Aldrich, St. Louis, MO, USA).

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Manufacturer protocol

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