Q5® Site-Directed Mutagenesis Kit

Site Directed Mutagenesis (SDM) Monkey - Deletion COS-7 PNPLA7

Experiment
Site Directed Mutagenesis (SDM) Monkey - Deletion COS-7 PNPLA7
Product
Q5® Site-Directed Mutagenesis Kit from New England BioLabs
Manufacturer
New England BioLabs

Protocol tips

Protocol tips
- Only use 1 μl of PCR product in the KLD reaction. Carrying too much PCR product forward can decrease transformation efficiency.

- Only use 5 μl of the KLD reaction in the transformation. If more KLD reaction is added, a buffer exchange step, such as PCR
purification, should be included prior to transformation.

Publication protocol

Plasmids and cloning of recombinant proteins
The vectors pDsRed2-ER, pmCherry-N1, and pEGFP-N3 were obtained from Clontech. pEGFP-N1 containing the coding sequence (cds) of human PLIN2 was a kind gift from Stefan Höning (Cologne, Germany). To generate a plasmid encoding PLIN2-mCherry, the cds of PLIN2 was amplified using the primers 5′-GCG AAT TCG CCA TGG CAT CCG TTG CAG TTG A-3′ and 5′-CAA CCG GTC GAT GAG TTT TAT GCT CAG ATC G-3′, and the PCR product was digested with EcoRI and AgeI and ligated into the multiple cloning site of pmCherry-N1. The cds of PNPLA7 was amplified by PCR using cDNA of murine cardiac muscle as template and the primers 5′-TTC TCG AGG CCA TGG AGG AGC AGT CCC AGT CC-3′ (F1) and 5′-GCG AAT TCG ACG AAG GAT GTT CCA GTC TTG G-3′ (R1). PNPLA7 truncation mutants were generated by PCR using the primers 5′-TTC TCG AGG CCA TGG CAG AAC CCA CTC CTC AGT AC-3′ (F2) and R1 for ΔTM-PNPLA7 (Δ1–40), 5′-TTC TCG AGG CCA TGG TTG TAA CGC GAC TGA TTC ATC TC-3′ (F3) and R1 for PNPLA7-C (Δ1–680), 5′-GCG AAT TCG CCA TGG AGG AGC AGT CCC AGT C-3′ (F4) and 5′-CAG GAT CCC CGT CGT AAT CTT CTC ACT C-3′ (R2) for TM-PNPLA7 (Δ41–1326), F1 and 5′-GCG AAT TCG ACT GTG GGT ACC TGC GCT TGA TAG-3′ (R3) for PNPLA7-N (Δ681–1326), F3 and 5′-GCG AAT TCG ATC TTG CCA CAT CCG CTG GGA G-3′ (R4) for PNPLA7-C1 (Δ1–680, Δ1091–1326), F3 and 5′-GCG AAT TCG AGA AGA TGT TGC TGA TGC TGC-3′ (R5) for PNPLA7-C2 (Δ1–680, Δ1018–1326), F3 and 5′-GCG AAT TCG AAG CAA ACA GAG CAC CCA TGA A-3′ (R6) for PNPLA7-C3 (Δ1–680, Δ968–1326), F3 and 5′-GCG AAT TCG AGG CAC ACC CTC TAG CTC CAC C-3′ (R7) for PNPLA7-C4 (Δ1–680, Δ936–1326), 5′-TTC TCG AGG CCA TGT TTG CTC TGG AGC TCC AAC A-3′ (F4) and R1 for PNPLA7-C5 (Δ1–741), 5′-TTC TCG AGG CCA TGT TGG TGC TTG GAG GGG GTG GA-3′ (F5) and R1 for PNPLA7-C6 (Δ1–923), 5′-TTC TCG AGG CCA TGT CCA TGG GGG CAA AGG TTG TG-3′ (F6) and R1 for PNPLA7-C7 (Δ1–1090), and F4 and R6 for PNPLA7-C8 (Δ1–741, Δ968–1326). The PCR products were purified by agarose gel electrophoresis and inserted into the multiple cloning site of pEGFP-N3 using the XhoI and EcoRI restriction sites to create C-terminal in-frame fusions with EGFP. Internal deletion mutants of PNPLA7 were generated using the Q5® site-directed mutagenesis kit with PNPLA7-EGFP as template and the primers 5′-CCG TCG TAA TCT TCT CAC-3′ and 5′-GTT GTA ACG CGA CTG ATT C-3′ for ΔR-PNPLA7 (Δ41–680), 5′-CAG ATC ATC ATG GTC CGG-3′ and 5′-CCG AAC ATT TTT CAA CAT GTA C-3′ for ΔCBD1 (Δ144–265), 5′-GTC CTG GGC GTG GCA CAC-3′ and 5′-GTC CTT TGT GGC AGC TCT GAA G-3′ for ΔCBD2 (Δ455–565), and 5′-GTT GTA ACG CGA CTG ATT C-3′ and 5′-CAT CCT CTT CAC AAC TGT G-3′ for ΔCBD3 (Δ578–680). A DNA fragment encoding for PNPLA7 harboring an N-terminal HA tag was created by PCR using the primers 5′-TAA TCT CGA GGC CAC CAT GTA CCC ATA CGA TGT TCC AGA TTA CGC TAT GGA GGA GCA GTC CCA GTC-3′ and 5′-GAC GAA TTC GTC AGG AAG GAT GTT CCA GTC-3′. The PCR product was purified as described above and inserted into the multiple cloning site of pEGFP-N3 using the XhoI and EcoRI restriction sites. To generate lentiviral expression vectors, pEGFP-N3 constructs encoding the open reading frames for EGFP, PNPLA7-EGFP, and PNPLA7-C-EGFP were digested with XhoI and NotI, and the resulting fragments were inserted into the multiple cloning site of pLVX IRES Puro (Clontech). Mission® lentiviral pLKO.1 vectors encoding for scrambled shRNA or shRNAs targeting murine PNPLA7 were obtained from Sigma-Aldrich. Constructs used for RNAi experiments targeted the sequences CCAAGAGGATTCTGCGCTTTA (shRNA1) and CATGTCCTTGTCAGGCTATAT (shRNA2) of the PNPLA7 cds.

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Papers

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Paper title
The phospholipase PNPLA7 functions as a lysophosphatidylcholine hydrolase and interacts with lipid droplets through its catalytic domain
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Manufacturer protocol

Download the product protocol from New England BioLabs for Q5® Site-Directed Mutagenesis Kit below.

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