Plasmids. The hDAGLa plasmid was constructed as described before.[38] Briefly, full length human cDNA of hDAGL-a was pur- chased from Biosource and cloned into mammalian expression vector pcDNA3.1, containing genes for ampicillin and neomycin re- sistance. A FLAG-linker was made from primers and cloned into the vector at the C-terminus of hDAGL-a. The plasmid was grown in XL-10 Z-competent cells and prepped (Maxi Prep, Qiagen). The sequences were confirmed by sequence analysis at the Leiden Genome Technology Centre. Transfection. HEK293T cells were grown to & 70 % confluency in 15 cm dishes. Prior to transfection, culture medium was refreshed (15 mL). A 3:1 (m:m) mixture of polyethyleneimine (PEI, 60 mg/well) and plasmid DNA (20 mg/well) was prepared in serum free culture medium and incubated for 10 min at rt. Transfection was per- formed by dropwise addition of the PEI/DNA mixture (2 mL/well) to the cells. 24 h post-transfection, the medium was refreshed and after 48 h cells were harvested. U2OS_ABHD6-GFP stable expression. Full-length human cDNA of ABHD6 (Source Bioscience) was cloned into mammalian expression vector pcDNA3.1, containing genes for ampicillin and neomycin re- sistance. The inserts were cloned in frame with a C-terminal FLAG- and GFP-tag. Plasmids were isolated from transformed XL-10 Z- competent cells (Maxi Prep kit: QiaGen) and sequenced at the Leiden Genome Technology Center. Sequences were analyzed and verified (CLC Main Workbench). One day prior to transfection U2OS cells were seeded to a 6 xxxxx plates (& 0.5 million cells/well). Prior to transfection, culture medium was aspirated and a minimal amount of medium was added. A 3:1 (m m@1) mixture of polyethy- leneimine (PEI) (3.75 mg/well) and plasmid DNA (11.25 mg/well) was prepared in serum free culture medium and incubated (15 min, RT). Transfection was performed by dropwise addition of the PEI/ DNA mixture to the cells. After 24 h, transfection efficiency was de- termined by fluorescence microscopy and transfection medium was exchanged for selection medium containing 800 mg mL@1 G418. 48 h Post-transfection single cells were seeded to 96 xxxxx plates in 100 mL selection medium. After 14 days, plates were in- spected for cell growth, clones were checked for ABHD6-GFP ex- pression by fluorescence microscopy (GFP channel). From here on, cells were grown in maintenance medium containing 400 mg mL@1 G418, and expanded in 12- and 6-xxxxx plates and 10 cm dishes. Inhibitor treatment. The medium was ...
Plasmids. Unless otherwise agreed in writing by the Parties, Client will notify OXB in writing which of the following will apply to the Manufacture of a Vector:
Plasmids. Unless otherwise agreed in writing by the Parties, Client will notify OXB in writing which of the following will apply to the Manufacture of a Vector:
(a) Plasmids incorporating a GOI (genome/transfer Plasmid) suitable for Manufacturing Vector shall be procured and provided to OXB by Client at Client’s sole cost;
(b) Plasmids incorporating a GOI (genome/transfer Plasmid) shall be procured by OXB on behalf of Client from an Approved Subcontractor and will be deemed Client Materials after Client’s payment therefor. [***]Pass-Through Costs relating to the manufacture of such Plasmids shall be charged by OXB to Client, such costs to include the direct cost of Plasmids, their transport and storage, and any import duties. In addition to such Pass-Through Costs, Client shall pay OXB a handling fee for procuring such Plasmids, which shall be [***] of such external costs and shall cover all OXB’s internal and other external costs associated with procuring such Plasmids (including stability testing performed by OXB). In such circumstances, OXB will notify Client in writing of the total costs for such Plasmids in advance of them being incurred. OXB will invoice Client following receipt by OXB of an applicable invoice from the Plasmid manufacturer. Title to such Plasmids shall pass to Client upon Client’s payment to OXB of the Pass-Through Costs (including the handling fee) for such Plasmids, and OXB shall inspect, store and handle such Plasmids in accordance with the provisions of this Agreement governing Client Materials. Notwithstanding any other provision of this Agreement, OXB accepts no liability for the performance of the Plasmid manufacturer under this sub-clause 3.8(b) however the Plasmids are sourced and in particular, in the event the Plasmid manufacturer fails to perform or delivers defective Plasmids to OXB, other than to the extent resulting from OXB’s gross negligence or wilful misconduct; or
(c) Helper Plasmids, i.e. env, rev and gag-pol Plasmids, shall be procured by OXB from an Approved Subcontractor at OXB’s sole cost and will be deemed Components.
Plasmids pBK-CMV expression vector. pMIGR1-eGFP Figure 2.1 plasmid maps of pBK-CMV and MIGR1
Plasmids. Table 2-4 Plasmids used in this study Name Cloning vector Insert Use Reference Table 2-5 Plasmids generated in this study Name Cloning vector Insert Generation of insert 5’ cloning site 3’ cloning site
