SgfI R7103

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	The amplified ORF and the pTUNE inducible vector (OriGene Technologies, Inc.) were digested with endonuclease XhoI (New England Biolabs, Inc., Beijing, China) and SgfI (Promega Corporation, Madison, WI, USA). The CDKN2A/p16-wild-type ORF was subsequently digested by the restriction enzymes XhoI and SgfI to construct the pTUNE-CDKN2A/p16-wild-type inducible vector. The positive clone was also sequenced for verification.

Protocol tips

The amplified ORF and the pTUNE inducible vector (OriGene Technologies, Inc.) were digested with endonuclease XhoI (New England Biolabs, Inc., Beijing, China) and SgfI (Promega Corporation, Madison, WI, USA). The CDKN2A/p16-wild-type ORF was subsequently digested by the restriction enzymes XhoI and SgfI to construct the pTUNE-CDKN2A/p16-wild-type inducible vector. The positive clone was also sequenced for verification.

Publication protocol

"The pTUNE inducible vector was obtained from OriGene Technologies Inc. (Rockville, MD, USA) as control vector. The primers specific for amplifying CDKN2A/p16 open reading frame (ORF) were synthesized by Invitrogen Life Technologies as follows: Primer 1 (hCDKN2A-SgfI), 5′-GAGGCGATGAGGCGATC GCATGGAGCCGGCGGCG-3′; primer 2 (hCDKN2A-XhoI), 5′-GCGCTCGAGATCGGGGATGTCTGAGGGACCTTC-3′; and primer 3 (hCDKN2A-XhoI), 5′-GCGCTCGAGAT CGGGGATGTCTGAGGGACCTTCCGCGGCATCTAG-3′.

The CDKN2A/p16-A148T ORF fragment was cloned from the cDNA template of UACC-1598 cells by polymerase chain reaction (PCR) with primers 1 and 2 specific to CDKN2A/p16. The PCR product was electrophoresed on a 2% agarose gel (Gene Tech Company Limited, Shanghai, China)and the predicted 490 bp fragment was extracted. The amplified ORF and the pTUNE inducible vector (OriGene Technologies, Inc.) were digested with endonuclease XhoI (New England Biolabs, Inc., Beijing, China) and SgfI (Promega Corporation, Madison, WI, USA). A ligation reaction was set up with linearized pTUNE vector and CDKN2A/p16 ORF using T4 DNA ligase (TransGen Biotech Co., Ltd., Beijing, China). The positive clone was picked up and the authenticity of the plasmid was confirmed by sequencing.

The CDKN2A/p16-wild-type ORF fragment was amplifed with primers 1 and 3 using the pTUNE-CDKN2A/p16-A148T vector template, which covers the mutation site and contains the wild-type base G substitution for correcting the sequence. The PCR product was separated by electrophoresis and the 490 bp fragment was extracted. The CDKN2A/p16-wild-type ORF was subsequently digested by the restriction enzymes XhoI and SgfI to construct the pTUNE-CDKN2A/p16-wild-type inducible vector. The positive clone was also sequenced for verification."

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