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. 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 pBK-CMV expression vector. Expresses the native untagged cDNA of WFDC1 in mammalian cells (A kind gift of Xxxxx Xxxxxx, Xxxxxxxxxx, USA) pMIGR1-eGFP Retroviral plasmid pMIGR1-eGFP encodes a multiple cloning site (MCS) upstream of enhanced green fluorescent protein (eGFP) separated by an internal ribosome entry site (IRES). The gene of interest is cloned into the MCS and the IRES ensures bicistronic expression of the eGFP reporter gene independently of the gene of interest. 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 pBluescript/ARS1 WTA N/A N/A Template for in vitro DNA replication assay (Marahrens and Xxxxxxxx, 1992) pJY22 pBS KS+ ARS1 Template for in vitro DNA replication assay (Xxxxxx et al., 2017) pET28a N/A N/A Construction of expression strains in E. coli Merck pJY19 pET28a PCNA Expression of PCNA in E. coli (Xxxxxx et al., 2017) pAM3 pGEX-6p-1 CDC6 Expression of Cdc6 in E. coli (Xxxxxxx et al., 2013) pFJD5 pFJD12 6HIS-GINS Expression of GINS in E. coli (Xxxxxx et al., 2009) pMD132 pBP6 HIS- MCM10- FLAG Expression of MCM10 in E. coli (Xxxxxxx et al., 2018) pCFK1 pGEX-6p-1 NAP1 Expression of NAP1 in E. coli (Xxxxx et al., 2017) N/A pCDFDuet Yeast histones H2A and H2B Expression of yeast histones H2A and H2B in E. coli (Kingston et al., 2011) N/A pETDuet Yeast histones H3 and H4 Expression of yeast histones H3 and H4 in E. coli (Xxxxxxxx et al., 2011) pRJ1228 pET28a NHP6 Expression of Nhp6 in E. coli (Xxxxx et al., 2003) N/A pRS303 FEN1-TEV- CBP Galactose- inducible expression in yeast Generated within the laboratory by Dr Anne Early N/A pRS303 CBP-TEV- DNA2 Galactose- inducible expression in yeast Generated within the laboratory by Dr Anne Early N/A pRS303 Lig1- 2XFLAG Galactose- inducible expression in yeast Generated within the laboratory by Xx Xxx Xxxxxx Table 2-5 Plasmids generated in this study Name Cloning vector Insert Generation of insert 5’ cloning site 3’ cloning site pJHH1 N/A CAC2 GeneArt Gene Synthesis AscI XhoI pJHH2 N/A CBP-CAC3 GeneArt Gene Synthesis SgrAI NotI pJHH3 N/A CBP- RTT106 GeneArt Gene Synthesis SgrAI NotI pJHH4 N/A CBP- VPS75 GeneArt Gene Synthesis SgrAI NotI pJHH6 N/A ASF1-CBP GeneArt Gene Synthesis SgrAI NotI pJHH7 N/A CAC1 GeneArt Gene Synthesis SgrAI NotI pJHH8 pRS306 CAC1 and CAC2 GeneArt Gene Synthesis SgrAI (CAC1) AscI (CAC2) NotI (CAC1) XhoI (CAC2) pJHH9 pRS303 CBP-CAC3 GeneArt Gene Synthesis SgrAI NotI pJHH10 pRS303 ASF1-CBP GeneArt Gene Synthesis SgrAI NotI pJHH11 pRS303 CBP- VPS75 GeneArt Gene Synthesis SgrAI NotI pJHH12 pRS303 CBP- Rtt106 GeneArt Gene Synthesis SgrAI NotI pJHH14 pET28a 6XHIS- Rtt109 PCR NdeI SalI pJHH16 pET28a 6XHIS- Pif1 PCR NheI NotI pJHH17 pETDuet 2XMyc-H3 and H4 JHH40 and JHH41 (Sigma) NcoI NcoI
