pCMV-PE2 (Addgene #132775, a gift from David Liu1 (link)) was digested with EcoRI-HF (NEB). After digestion, the plasmid was dephosphorylated by rSAP (NEB), followed by gel extraction. tagRFP was PCR amplified from pLV312.3 (Addgene #119944)19 (link). tagRFP was Gibson assembled into pCMV-PE2 using 50 ng backbone and 2:1 ratio of tagRFP amplicon. Assembly was performed with NEBuilder HiFi DNA Assembly Master Mix (NEB). Assembled plasmids were transformed into NEB Stable Competent E. coli, resulting in pCMV-PE2-tagRFP. P2A-BleoR (Zeocin resistance) was PCR amplified from an in-house plasmid that originated from pcDNA™3.1/Zeo (Invitrogen). pCMV-PE2-tagRFP was PCR amplified to get linearized plasmid, with amplicon end downstream of tagRFP. Gibson assembly was performed as described previously, resulting in pCMV-PE2-tagRFP-BleoR (Addgene #192508). Lenti-p3-eGFP plasmid was produced by replacing EF1a-PuroR (MluI-HF and ApaI) on Lenti-gRNA-puro (Addgene #84752, a gift from Hyongbum Kim20 (link)) with p3-eGFP sequence.
Protocol detail
Construction of pCMV-PE2-tagRFP-BleoR Plasmid
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Other organizations : University of Zurich
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Variable analysis
independent variables
- Digestion of pCMV-PE2 plasmid with EcoRI-HF
- Dephosphorylation of plasmid by rSAP
- PCR amplification of tagRFP from pLV312.3
- Gibson assembly of tagRFP into pCMV-PE2
- PCR amplification of P2A-BleoR from an in-house plasmid
- Gibson assembly of P2A-BleoR into pCMV-PE2-tagRFP
- Replacement of EF1a-PuroR with p3-eGFP sequence on Lenti-gRNA-puro plasmid
- Successful cloning and construction of pCMV-PE2-tagRFP and pCMV-PE2-tagRFP-BleoR plasmids
- Successful construction of Lenti-p3-eGFP plasmid
- Competent E. coli strain used for transformation (NEB Stable Competent E. coli)
- NEBuilder HiFi DNA Assembly Master Mix used for Gibson assembly
- Addgene #132775 (pCMV-PE2) as a source for the backbone plasmid
- Addgene #119944 (pLV312.3) as a source for the tagRFP sequence
- In-house plasmid containing P2A-BleoR sequence
- None explicitly mentioned
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