Modified wild type (WT) and mutant pUNO-CARD11-FLAG plasmids were constructed and purified as previously described (9 (link)). Briefly, site-directed mutagenesis was utilized to introduce single nucleotide variants that were putative point mutations into the WT CARD11 construct (Invivogen) using primer-directed linear amplification with Pwo or Pful polymerase (Roche), followed by Dpnl digestion of methylated template DNA (ThermoFisher). All inserted variants were confirmed by Sanger sequencing. All resulting plasmids were purified from DH5α E. Coli (New England Biolabs) using a GenElute HP Plasmid Maxi-Prep Kit (Sigma).
Genelute hp plasmid maxi prep kit
Manufactured by Merck Group
Sourced in United States
The GenElute HP Plasmid Maxi-Prep Kit is a laboratory product designed for the purification of high-quality plasmid DNA from bacterial cultures. It utilizes a silica-based membrane technology to efficiently capture and purify plasmid DNA from cell lysates.
Automatically generated - may contain errors
Lab products found in correlation
E coli dh5α, by New England Biolabs (3 mentions)
Attractene transfection reagent, by Qiagen (3 mentions)
Blastidicin, by InvivoGen (2 mentions)
Human puno card11 plasmid, by InvivoGen (2 mentions)
Rapid dephosphorylation ligation kit, by Roche (2 mentions)
Pwo dna polymerase, by Roche (2 mentions)
Pbabe neo, by Addgene (1 mentions)
Pegfp c1 er alpha, by Addgene (1 mentions)
Xhoi, by New England Biolabs (1 mentions)
Genelute plasmid miniprep kit, by Merck Group (1 mentions)
Pegfp n1 expression vector, by Takara Bio (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Rpmi 1640, by Merck Group (1 mentions)
Trypan blue, by Merck Group (1 mentions)
L dopa, by Merck Group (1 mentions)
Histopaque 1077, by Merck Group (1 mentions)
Neocuproine, by Merck Group (1 mentions)
Bathocuproine, by Merck Group (1 mentions)
Pfuse higg1 fc2, by InvivoGen (1 mentions)
Optima l 90k ultracentrifuge, by Beckman Coulter (1 mentions)
14 protocols using genelute hp plasmid maxi prep kit
1
CARD11 Mutant Plasmid Construction
2
Plasmid Transfection Protocol for MDA-MB-231 Cells
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The ER(α) expression plasmid pEGFP-C1-ER alpha, ER(β) expression plasmid pCDNA3.1-nv5-ER beta and scramble vector Pbabe-neo were purchased from Addgene (Addgene plasmids #28,230, #22,770, and #1767, respectively). After being transformed using the heat shock technique, the Escherichia coli DH5α strain was spread using a sterile loop onto a prepared lysogeny broth (LB) agar plate containing kanamycin or ampicillin respectively, to isolate individual colonies of bacteria carrying the plasmids cited above and incubated overnight at 37 C. After 24 h, one colony was transferred into LB media with the corresponding antibiotic and incubated at 37 C for while shaking. After incubation, bacterial growth was characterized by a cloudy haze in the media. The plasmids were extracted and purified from the transformed and proliferated Escherichia coli DH5α using the GenElute HP Plasmid Maxiprep kit (Sigma‒Aldrich). MDA-MB-231 cells were then transfected using the traditional protocol with Attractene Transfection Reagent (Qiagen) following the manufacturer’s instructions. After 48 h of transfection, RNA and proteins were extracted as previously described.
3
Isolation and Purification of scFv DNA
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The double strand DNA plasmids containing the scFvs were isolated from each cycle of selection from a culture of superinfected E. coli TG1 cells using GenElute HP Plasmid Maxiprep Kit (Sigma-Aldrich). The VHs were excised by double digestion with restriction enzymes NcoI and XhoI (New England Biolabs) and then purified from a 1.2% agarose gel (Figure 1(a) ).
4
Plasmid Transfection of hTERT Variants
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Wild-type hTERT (hTERT-WT) and dominant-negative hTERT mutant (hTERT-DN) expression plasmids pBabe-neo-hTERT and pBabe-puro-DN-hTERT, respectively, were a gift from Bob Weinberg (Addgene plasmid #1774 and #1775) [35 (link)], while the empty vector pBabe-neo, was a gift from Hartmut Land & Jay Morgenstern & Bob Weinberg (Addgene plasmid # 1767) [36 (link)]. Plasmids were purified from the transformed E.coli DH5alpha bacteria with a GenElute Plasmid Miniprep Kit and then with a GenElute HP Plasmid Maxiprep kit (Sigma-Aldrich, USA). Cells were then transfected using the fast-forward protocol with Attractene Transfection Reagent (Qiagen Inc.) for 24h and 48h, according to the manufacturer’s instructions.
5
Engineering Recombinant NDV-GFP Virus
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The NDVF3aa-GFP genome was engineered to include an amino acid substitution in the fusion protein, of the 289th amino acid from leucine to alanine. Second, NDV was made to express the full-length enhanced GFP protein, with the GFP gene located between the phosphoprotein (P) and matrix (M) genes of NDV. The helper plasmids (pTM1-NP, pTM1-P, and pTM1-L) were a kind gift from Dr. Peter Palese (Mount Sinai, NY, USA). Plasmids were purified with the GenElute HP Plasmid Maxiprep Kit (Sigma-Aldrich). Recombinant NDV-GFP was rescued and propagated in specific pathogen-free eggs (Canadian Food and Inspection Agency), and allantoic fluid was harvested 50 h post-inoculation and clarified by centrifugation (1,500 × g for 10 min at 4°C) and purified as described.93 (link) The virus was aliquoted and stored at −80°C and subsequently referred to as “NDV.”
6
Truncated VP1 Variants Production
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The codon-optimized VP1- and VP2-coding plasmids used were pwM (Addgene plasmid 22515) and ph2m (Addgene plasmid 22518), respectively (sequences from MCC isolate 339 [5 (link)]).
The C-terminally truncated VP1 variants VP11–384 and VP11–402 were produced inEscherichia coli DH5α cells by introducing additional ochre stop codons (UAA) into the pwM plasmid using site-directed mutagenesis. All plasmids were propagated in E. coli XL10-Gold cells and purified for mammalian cell transfection using the GenElute HP plasmid maxiprep kit (Sigma-Aldrich).
The C-terminally truncated VP1 variants VP11–384 and VP11–402 were produced in
7
Plasmid DNA Isolation and Mutagenesis
8
Overexpression of hTERT in Cancer Cell Lines
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The three cell lines were transfected with two types of vectors, pBabe-neo-hTERT (cat. no. 1774) and pBabe-neo (control; cat. no. 1767), gifted from Addgene, Inc. After plasmid purification from transformed bacteria using a GenElute HP Plasmid Maxiprep kit (Sigma-Aldrich; Merck KGaA), the transfection was performed using Attractene Transfection Reagent (Qiagen Inc.), according to the manufacturer's protocols. Briefly, the cells were seeded in 6-well plates at a density of 0.5×106 cells/well. Prior to seeding, 1.2 µg of plasmid, attractene and serum-free DMEM (4.5 g/l glucose; for SKOV-3 and Igrov-1), or F-12 for Ovcar-3, were mixed in the wells and incubated for 15 min at room temperature. Subsequently, cells were seeded in 6-well plates for 48 h at 37°C prior to subsequent experiments.
9
Plasmid Isolation and Catecholamine Preparation
10
Generation and Validation of Modified CARD11 Constructs
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The human pUNO-CARD11 plasmid (Invivogen) was modified to include a 3’ 3X FLAG tag using annealed, overlapping oligonucleotides encoding the tag and inserted via BamHI (5’) and NheI (3’) overhangs. Single point mutations were introduced into the WT CARD11 construct by site-directed mutagenesis, using specific primers for linear amplification using 2x Pwo DNA polymerase (Roche) and subsequent digestion with DpnI to destroy methylated template DNA (ThermoFisher Scientific). A 42 bp fragment encoding aa183–196 duplication was generated by overlap extension PCR and ligated into a BsrG1 site in pUNO-CARD11 using a rapid dephosphorylation/ligation kit (Roche). Mutations were confirmed by Sanger sequencing. All plasmids were purified using a GenElute HP Plasmid Maxi-Prep Kit (Sigma) from transformed competent DH5α E. coli (New England Biolabs) selected with blastidicin (InvivoGen).
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