Tyrosine to phenylalanine (Y>F) ASC mutants of the pEF6-ASC-GFP plasmid were generated using the Q5 site-directed mutagenesis kit (New England Biolabs) according to the manufacturer's instructions. The mutated plasmids were transformed into DH5α chemically competent E. coli and then isolated by mini- or midi-preps (Qiagen). The isolated plasmids were then sequenced to validate the mutations.
Midi preps
Manufactured by Qiagen
The QIAGEN Midi preps are a laboratory equipment product designed for the purification of plasmid DNA from bacterial cultures. The core function of this product is to provide a reliable and efficient method for extracting and purifying plasmid DNA samples in a mid-scale range.
Automatically generated - may contain errors
Lab products found in correlation
Q5 site directed mutagenesis kit, by New England Biolabs (1 mentions)
Minelute pcr purification kit, by Qiagen (1 mentions)
Nebuilder hifi dna assembly master mix, by New England Biolabs (1 mentions)
Bamhi, by New England Biolabs (1 mentions)
Rapid alkaline phosphatase, by Roche (1 mentions)
Neb stable competent escherichia coli high efficiency, by New England Biolabs (1 mentions)
Zymoclean large fragment dna recovery kit, by Zymo Research (1 mentions)
Gel purification, by Qiagen (1 mentions)
P3xflag cmv 7.1 vector, by Merck Group (1 mentions)
Pgex 6p 2, by Cytiva (1 mentions)
Pcmv myc, by BD (1 mentions)
Sanger sequencing, by GenScript (1 mentions)
5 protocols using midi preps
1
Tyrosine to Phenylalanine ASC Mutants
2
Sawfly Silk Protein Expression
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Example 14
The introduction of restriction enzyme digestion sites into the sawfly DNA isolate of SEQ ID No: 23 allowed isolation of the DNA for a sawfly silk gene and its insertion into an expression vector. Sawfly collagen-like silk type A gene was inserted into pColdI vector via NdeI and EcoRI sites. The PCR colony screening technique was used to detect positive clones. These clones were grown up in 100 ml culture volumes and Qiagen midi preps carried out to expand the vector quantity. For expression, a selected positive clone was transformed into competent E. coli BL21 cells. For expression of the sawfly silk protein gene, one colony of cells was added to 100 ml starter culture medium, 2× YT-Amp and incubated at 37° C. with 200 rpm shaking overnight. This culture then had 100 ml fresh 2×YT-2% Glucose-Amp added, and was induced with 1 mM IPTG at 25° C. for 10 hour, then 20° C. for another 16 hour. The cell paste was harvested by centrifugation (3000×g for 30 min). The protein was associated with the cell pellet.
3
Construction of Chimeric Antigen Receptor Plasmids
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The different constructs, as shown in Figure 1 , were generated by Gibson Assembly. The two CAR backbone constructs were ordered as gBlock (IDT, Leuven, Belgium) and cloned into the LZRS-IRES-eGFP retroviral plasmid by Gibson Assembly (NEBuilder HiFi DNA Assembly Master Mix, NEB, Ipswitch, MA, USA). The nanobody specific sequences were amplified using PCR. PCR products were visualized on gel and purified using the MinElute PCR Purification kit (Qiagen, Venlo, The Netherlands) as per fabricator instructions. The LZRS-CAR-IRES-eGFP plasmids were overnight digested with BamHI (NEB) and purified using the Zymoclean Large Fragment DNA Recovery Kit (Zymo, Irvine, CA, USA) according to manufacture instructions. Subsequently, digested and purified plasmid was dephosphorylated (rAPid Alkaline Phosphatase, Roche, Vilvoorde, Belgium) and used in a Gibson Assembly reaction together with the purified PCR products. The Gibson Assembly reaction mix was transformed in bacteria (NEB Stable Competent Escherichia coli (High Efficiency), NEB) and plated on agar. After overnight incubation, colonies were selected and grown in liquid lysogenic broth (BD Difco, Erebodegem, Belgium) overnight. Plasmids were isolated and sequenced. Colonies containing the correct plasmid were further cultured and midipreps (Qiagen) were performed.
4
FOXQ1 and RbBP5 Construct Generation
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For the generation of FOXQ1 fragment constructs, wild-type pENTR-FOXQ1 was used as the DNA template. FOXQ1 was subcloned into GST vector, pGEx-6p2 (Amersham Biosciences, 18-1157-58), with GST fused to the N-terminal by PCR with 5′ BamHI and 3′ XhoI restriction sequences. FOXQ1 fragments were cloned into P3XFLAG-CMV 7.1 vector (Sigma-Aldrich, E7533) with N-terminal FLAG-epitope tag with 5′ HindIII and 3′ KpnI endonuclease sequences. RbBP5 plasmid was purchased from Addgene (#15550). RbBP5 regions were cloned into pCMV-MYC (BD Biosciences, K6003-1) by PCR with N-terminal MYC-epitope tag with 5′ EcoRI and 3′ KpnI sequences. PCR products were purified by gel purification (Qiagen) and subjected to double digestion overnight at 37 °C. The double digestion product was purified by gel purification. The ligation reaction was performed with a 5:1 insert to vector molar ratio with T4 DNA ligase and incubated at 16 °C overnight. Five μL ligation reaction was heat shock transformed into 25 μL DH5α competent cells. Clones were picked from single colonies. DNA plasmids were purified by Qiagen mini and midi preps according to the manufacturer’s protocol. Plasmids were validated through double digestion and Sanger sequencing (GenScript).
5
Casp Protein Purification and Cell Induction
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Casp was procured from DGRC (FMO05904), which had C-terminal FLAG and 6X-His tags and also subcloned into pRM-HA3 both untagged and also with a N-terminal 6XHis tag. All constructs were sequenced after subcloning and before use. QIAGEN Midipreps were used to generate plasmid DNA (1 μg/μL) that was transfected into cells using Mirus TransIT transfection agent based on the manufacturer’s recommendations. Cell lysis, FLAG or HA affinity, SDS-PAGE gels, IPs, and Western blots were carried out or processed as per protocols described earlier in this section. For Casp IP experiments, cells were transfected in a single flask at 50% confluency, and 0.5 mM CuSO4 was added on the same day. After 48 hr, allowing for Casp expression, equal number of cells were split into a 12-well plate, 0.5 mL cells per well, allowing for multiple experiments at the same transfection efficiency. Cells (529SU or S2) were then heat shocked (1 hr, 37°, or treated with LPS (10 μg/mL; 1−6 hr). For the Casp/LPS induction experiments with 529SU cells, equal amount of protein, as measured by the Bradford assay was loaded into each well.
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