To overexpress either C-terminal Myc-tagged or GFP-tagged PRMT7, pLenti-C-MycDDK or pLenti-C-mGFP, respectively, were purchased from Origene. A control vector expressing only GFP or Myc was created via blunt-end digestion and subsequent ligation of the pLenti-C-mGFP-PRMT7 and pLenti-C-MycDDK-PRMT7 vectors, respectively (characterized in Baldwin et al., 2015 (link)). Mutant eIF2α-GST protein from pGEX-4T2 vector was designed: R52R53R54 mutated to KKK, RKK, KRR, RKR, and RRK. Primers were designed using QuickChange Primer Design by Agilent Technologies. These constructs were then subcloned into the pLenti-C-mGFP backbone as described above. To knock down PRMT7, RNA interference was performed using pLKO.1 vectors obtained from The RNAi Consortium containing either an shRNA with a luciferase sequence for control (5′-CAAATCACAGAATCGTCGTAT-3′) or two independent PRMT7 sequences (5′-GCTAACCACTTGGAAGATAAA-3′ and 5′-CGATGACTACTGCGTATGGTA-3′).
Quickchange primer design
Manufactured by Agilent Technologies
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The QuickChange Primer Design is a software tool that enables the design of primers for site-directed mutagenesis experiments. The tool analyses DNA sequences and generates appropriate primer sequences that can be used to introduce specific mutations or changes in the target DNA.
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6 protocols using quickchange primer design
1
Overexpression and Knockdown of PRMT7
2
Site-directed Mutagenesis of CbDAE Enzyme
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Site-directed mutagenesis was performed via polymerase chain reaction (PCR) using the KOD plus mutagenesis kit (Toyobo, Tokyo, Japan) with the pET22b-CbDAE plasmid as the template. The primers were designed using QuickChange Primer Design (Agilent, United States) and are listed in Supplementary Table S1. After the PCR reaction was complete, the template was removed by digestion with DpnI, and the product was further cyclized by T4 polynucleotide kinase and Ligation High DNA ligase. The mutations were confirmed by DNA sequencing (AZENTA, Suzhou, China). The plasmids harboring the correct mutated sequences were introduced into E. coli BL21 (DE3) for expression of the mutant proteins.
3
Site Directed Mutagenesis of GDAP1 Gene
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Site directed mutagenesis was performed on the OriGene plasmid: pCMV6-XL5 with GDAP1 (NM_018972) Human Untagged Clone, using the Mut Express II Fast Mutagenesis Kit V2 (Vazyme) in accordance with the manufacturer’s instructions.
Primers for mutagenesis were designed using Quick Change Primer Design (Agilent) on-line software (the primer sequences were placed inTable A1 ).
Escherichia coli strain XL1-Blue was used for plasmid propagation. The presence of mutations within constructed plasmids was verified by the Sanger sequencing method.
Primers for mutagenesis were designed using Quick Change Primer Design (Agilent) on-line software (the primer sequences were placed in
Escherichia coli strain XL1-Blue was used for plasmid propagation. The presence of mutations within constructed plasmids was verified by the Sanger sequencing method.
4
Construction and Validation of UGT2B3(S316N) Mutant
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UGT2B3 exhibits 83% identity to UGT2B2 (Mackenzie, 1986 (link)) in amino acid sequences. The Asn 316 of UGT2B2 is a potential site for glycosylation. Therefore, the Ser 316 of UGT2B3 was replaced with Asn by site-directed mutagenesis (SDM), and an expression system for the UGT2B3(S316N) mutant was constructed in a similar way as described above. The primers for the SDM were designed by Quick Change Primer Design (Agilent Technology). The primers used were UGT2B3(931, 964)(947G→A)SDM-F, 5′-GGG TCA ATG GTC AGC AAC ATG ACA GAA GAA AAG G-3′ and UGT2B3(964, 931)(947C→T)SDM-R, 5′-CCT TTT CTT CTG TCA TGT TGC TGA CCA TTG ACC C-3′. The procedures were carried out according to the manufacturer’s recommendations. The introduction of the mutation at the appropriate position and the absence of other unwanted mutations were confirmed by DNA sequencing.
5
Generating Mutant Rap1 Plasmids
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All experiments are performed using BY4741/4742 background, and deletion strains are from the haploid yeast knockout collection or were constructed using standard gene replacement techniques. Plasmids were made using Gateway cloning methods. Site‐directed mutagenesis to generate Rap1 Escherichia coli expression plasmids with AAA mutations in the SANT domain was performed using QuickChange primer design (Agilent) and primer extension using Phusion HF to introduce the changes into pGST‐SANT‐6xHis (BSS48); all mutations were verified by sequencing. All strains and plasmids used are listed in Table S1 , and primers used for mutagenesis are listed in Table S2 .
6
Targeted silencing and overexpression of UPF1 and AREG
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A short hairpin RNA (shRNA) sequence specifically targeting UPF1 (Table SIV) was cloned into Phblv-U6-puro (provided by Professor Fan Handong, Hangzhou Normal University) by BamHI and EcoRI digestion. The siRNA targeting AREG (targeted sequence, CCACAAATACCTGGCTATA) and a scrambled sequence were purchased from Guangzhou RiboBio Co., Ltd. The UPF1 insA and UPF1 del plasmids were constructed based on the pCMV-MYC-UPF1 vector (provided by Professor Lynne E. Maquat, University of Rochester). Primers for site-directed mutagenesis (Table SIV) were designed by QuickChange Primer Design (Agilent Technologies, Inc.) and applied with the KOD-Plus-PCR enzyme (Toyobo Life Science). Residual templates were digested by DpnI (New England Biolabs, Inc.) at 37°C for 5 h. The AREG gene open reading frame (ORF) with or without the 3′ untranslated region (3′UTR), which was referred as AREG-3′UTR-pEGFP or AREG-ORF-pEGFP, respectively, was cloned into the pEGFP-N1 vector (provided by Dr Wang Miao, Hangzhou Normal University) using the HindIII and BamHI restriction sites. The primer sequences used are listed in Table SIV. The AREG 3′UTR sequence was amplified and cloned into the dual-luciferase reporter construct pEZX-FR02 (GeneCopoeia, Inc.) by double digestion with EcoRI and SpeI (the primer sequences used are listed in Table SIV) to generate the pEZX-AREG-3′UTR construct.
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