The Pi4kap2 coding sequence was amplified from pDONR223-PI4KAP2 (23601 Addgene) using primers (Supplemental Experimental Procedures ) that introduced an HA-tag at the 3′ end and flanked the fragment with PstI and AgeI restriction enzyme sequences. The fragment was ligated into the pJet cloning vector using CloneJET kit instructions (K1231 Thermo Fisher Scientific) and amplified in Stbl3 bacteria (C737303 Thermo Fisher Scientific). The pLV-Ef1a-MCS-IRES-RFP-puro plasmid and the Pi4kap2HA-pJet vector were linearized with PstI and AgeI. The Pi4kap2-HA fragment was gel purified and ligated into the pLV vector using T4 ligase. For the shRNA vector purchased from Origene (TL510615), the MND-GFP cassette (a gift from the Kohn Laboratory) was used to replace the CMV-GFP reporter.
Mnd gfp cassette
Manufactured by OriGene
The MND-GFP cassette is a plasmid-based construct that contains the MND promoter and the gene encoding green fluorescent protein (GFP). This cassette can be used for the expression of GFP in various cell types.
Automatically generated - may contain errors
2 protocols using mnd gfp cassette
1
Cloning and Ligation of Pi4kap2 into pLV Vector
2
Cloning and Ligation of Pi4kap2 into pLV Vector
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The Pi4kap2 coding sequence was amplified from pDONR223-PI4KAP2 (23601 Addgene) using primers (Supplemental Experimental Procedures ) that introduced an HA-tag at the 3′ end and flanked the fragment with PstI and AgeI restriction enzyme sequences. The fragment was ligated into the pJet cloning vector using CloneJET kit instructions (K1231 Thermo Fisher Scientific) and amplified in Stbl3 bacteria (C737303 Thermo Fisher Scientific). The pLV-Ef1a-MCS-IRES-RFP-puro plasmid and the Pi4kap2HA-pJet vector were linearized with PstI and AgeI. The Pi4kap2-HA fragment was gel purified and ligated into the pLV vector using T4 ligase. For the shRNA vector purchased from Origene (TL510615), the MND-GFP cassette (a gift from the Kohn Laboratory) was used to replace the CMV-GFP reporter.
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