The largest database of trusted experimental protocols

11 protocols using pcdna5 frt plasmid

1

Doxycycline-Inducible N2A Flp-In Stable Lines

Check if the same lab product or an alternative is used in the 5 most similar protocols
Doxycycline inducible N2A Flp-In rtTA3 stable cell lines were generated as previously described (Gonatopoulos-Pournatzis et al., 2018 (link)). Briefly, N2A Flp-In rtTA3 stable cell lines capable of inducible cDNA expression were created through co-transfection of 200 ng of a rtTA3 compatible pcDNA5/FRT plasmid (modified from Thermo Fisher Scientific, V6010–20), with 2 μg of pOG44 Flp-recombinase expression vector (Thermo Fisher Scientific, V600520). Cell lines with successful cDNA integration were selected and maintained using 200 mg/mL Hygromycin.
+ Open protocol
+ Expand
2

Cloning and Expression of Protein Constructs

Check if the same lab product or an alternative is used in the 5 most similar protocols
3xFLAG-tagged SANS full length and deletion constructs were designed and cloned in house as previously described (29 (link)). HA-PRPF31 constructs were kind gifts from Dr. Utz Fischer (41 (link)). Deletion constructs of PRPF31 (N-term (aa1–165), C-term (aa 166–499)) were produced by PCR from the HA-PRPF31 plasmid and cloned into the pcDNA3.1 N-HA vector (Thermo Fisher). Deletion constructs of PRPF6 (NTD (aa 1–126), HAT1-6 (aa 293–592), HAT7-13 (aa 593–941)) were cloned into AcGFP-C1 vector (Addgene: 64607). RON (MST1R) minigene was a kind gift from Dr. Julian Koenig (IMB-Mainz), and USH1C minigene was amplified from the genomic DNA of HEK293T cells and cloned into the pcDNA5/FRT vector (Thermo Fisher). The pcDNA5/FRT-PRPF4 plasmid was constructed by cloning the PRPF4 gene fused to an N-terminal FLAG/HA tag into the pcDNA5/FRT plasmid (Thermo Fisher).
+ Open protocol
+ Expand
3

Purification and Analysis of IgG1 Antibodies

Check if the same lab product or an alternative is used in the 5 most similar protocols
Abrin toxin was prepared by Biointron Biological Inc. (Beijing). CCK-8 detection kits were purchased from Dojindo (Japan). TNT® Coupled Reticulocyte Lysate Systems were purchased from Promega (Cat. No.: L4610). Dulbecco’s modified Eagle medium (DMEM) (Cat. No.: 11965-092), RPMI 1640 media (Cat. No.: 61870-036) and fetal bovine serum (FBS) (Cat. No.: 10438-034) were purchased from ThermoFisher (U.S.). pFRT-KIgG1, a full-IgG expression plasmid fused with the constant regions of IgG1 heavy chain and kappa chain, was prepared in our lab based on the pcDNA5-FRT plasmid (ThermoFisher, Cat. No.: V601020). ExpiCHO-S cells and ExpiCHO™ Expression System (Cat. No.: A29133) were purchased from Gibco (ThermoFisher, U.S.). HiTrap MabSelect SuRe was purchased from Cytiva (U.S.). Alexa-FluorTM 488 and 594 were prepared by Jiaxuan Biotech (Beijing, China). TruStain FcXTM (anti-mouse CD16/32 and anti-Human IgG Fc receptors) were procured from Biolegend. All plastic-ware was procured from NuncTM (Denmark). All other chemicals were from commercial sources and of analytical grade.
+ Open protocol
+ Expand
4

Molecular Cloning of Protein Constructs

Check if the same lab product or an alternative is used in the 5 most similar protocols
3xFLAG-tagged SANS full length and deletion constructs were designed and cloned in house as previously described (29) . HA-PRPF31 constructs were kind gifts from Dr. Utz Fischer (University of Wuerzburg, Germany). Deletion constructs of PRPF31 (N-term (aa1-165), Cterm (aa 166-499)) were produced by PCR from the HA-PRPF31 plasmid and cloned into the pcDNA3.1 N-HA vector (Thermo Fisher). Deletion constructs of PRPF6 (NTD (aa 1-126), HAT1-6 (aa 293-592), HAT7-13 (aa 593-941)) were cloned into AcGFP-C1 vector (Addgene: 64607). RON (MST1R) minigene was a kind gift from Dr. Julian Koenig (IMB-Mainz), and USH1C minigene was amplified from the genomic DNA of HEK293T cells and cloned into the pcDNA5/FRT vector (Thermo Fisher). The pcDNA5/FRT-PRPF4 plasmid was constructed by cloning the PRPF4 gene fused to an N-terminal FLAG/HA tag into the pcDNA5/FRT plasmid (Thermo Fisher).
+ Open protocol
+ Expand
5

Molecular Engineering of Fluorescent AQP3 and TMC1

Check if the same lab product or an alternative is used in the 5 most similar protocols
The sequence of turboGFP variant (Evrogen, Moscow, Russia) was fused to the C-terminal of human AQP3 (UniProtKB/Swiss-Prot #Q92482.2) in silico using Lasergene II software (DNASTAR, Madison, WI). The full cDNA containing AQP3-GFP was then synthesized by Genscript (Piscatawas, NJ). The cDNA was subcloned into a pcDNA5/FRT plasmid (Invitrogen, Carlsbad, CA) using restriction enzymes Hind3/EcoRV. Each murine TMC1 (Pubmed #NP_083229.1) construct was also based on custom synthesis by Genscript with its known ER retention motifs KK or RRR substituted for alanines, and codon optimized for mammalian expression in HEK293 cells. Each Tmc1 based sequence was then subcloned into the AQP3-GFP-pcDNA5/FRT plasmid using Hpa1/EcoRV restriction sites. The palmitoylated GFP was custom synthesized, codon optimized for mammalian HEK293 expression and subcloned into pcDNA5/FRT plasmid using Nhe1/EcoRV restriction sites by Genscript. All constructs were confirmed by sequencing.
To express TMC1-YFP construct, the C-terminus of coding region of mouse Tmc1 was fused to the coding sequence of mVenus reporter using Hind III and BamHI sites in an mVenus-N1 vector36 (link). Sequencing of this construct confirmed the identity as murine Tmc1 (Pumbed NM_028953).
+ Open protocol
+ Expand
6

Cloning and Expressing Human α1 and α2

Check if the same lab product or an alternative is used in the 5 most similar protocols
DNAs encoding full-length human α1 and α2 were amplified with primers designed to encode a 5′ Kpn1 site, and a 3′ FLAG-tag followed by an Xho1 site. The resulting PCR products were cloned into the pcDNA5/FRT plasmid (Invitrogen). Stable cell lines were generated and cultured as described previously [19 (link)].
+ Open protocol
+ Expand
7

Stable Cell Line Generation of Ndc80 Tail Mutants

Check if the same lab product or an alternative is used in the 5 most similar protocols
To create a stable cell line expressing the Ndc80WT, Ndc80+4CT and Ndc80+4NT tail mutant, appropriate sequences were cloned into the pCDNA5/FRT plasmid (Invitrogen) using flanking Not1 restriction sites. The plasmid was co-transfected into T-Rex HeLa cells (Invitrogen) with the pOG44 plasmid (Invitrogen) and the cells were cultured in DMEM + 10% FBS (Gibco) supplemented with hygromycin B (Invitrogen) for 14 days. At the end of the selection period, remaining cells were pooled and used for subsequent experiments.
+ Open protocol
+ Expand
8

Generation of AQP6-tGFP Fusion Constructs

Check if the same lab product or an alternative is used in the 5 most similar protocols
The AQP3-tGFP-pcDNA5/FRT plasmid was synthesized as previously described17 (link). The full cDNA containing human AQP6-tGFP (NP_001643) was synthesized by Biomatik (Wilmington, DE) and tGFP-AQP6 (rat NP_071517.1) by Genscript (Piscatawas, NJ). In the tGFP-AQP6 construct, the tGFP was separated from AQP6 by the following peptide linker which included a Precision Protease site (underlined in blue): AASAVNGSLEVLFQGPAA, and containing Afe1 and Hpa1 restriction sites, as well as a 10× C-terminal Histamine Tag (Histag) as shown in Fig. 1A (in black). For the AQP6-tGFP construct, the tGFP was separated from AQP6 by the following peptide linker (Fig. 1B in blue) which included a Precision Protease site: GGSLEVLFQGPAA and a c-terminal 10× Histag (in black) as shown in Fig. 1B.
The AQP6-tGFP construct was subcloned into a pcDNA5/FRT plasmid (Invitrogen, Carlsbad, CA) using restriction enzymes BamH1/EcoRV while the AQP6 was subcloned using Hpa1/EcoRI into a pcDNA5/FRT plasmid containing tGFP. Each construct was custom synthesized by Genscript, codon optimized for mammalian expression in HEK293 cells. Each AQP6-based sequence was then subcloned into the AQP3-tGFP-pcDNA5/FRT plasmid using Hpa1/EcoRV restriction sites. All constructs were confirmed by sequencing.
+ Open protocol
+ Expand
9

Generation of pDF_FRT Plasmid

Check if the same lab product or an alternative is used in the 5 most similar protocols
For generation of the integration plasmid pDF_FRT, eGFP and mCherry expressed from a shared CMV enhancer/promoter element (pBI-CMV1; Clontech), were inserted into the NotI and NheI sites of the pcDNA5/FRT plasmid (Invitrogen). pDF_FRT_UCP3_wt and _MUTI/II were generated by hybridization of complementary oligonucleotides and insertion downstream of eGFP into the NotI and HindIII restriction sites of pDF_FRT.
The complete vector sequences are available upon request.
+ Open protocol
+ Expand
10

Stable Expression of Human Alpha Subunits

Check if the same lab product or an alternative is used in the 5 most similar protocols
DNAs encoding full-length human α1 and α2 were amplified with primers designed to encode a 5′ KpnI site, and a 3′ FLAG-tag followed by an XhoI site. The resulting PCR products were cloned into the pcDNA5/FRT plasmid (Invitrogen). Stable cell lines were generated and cultured as described previously [19 (link)].
+ Open protocol
+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required

Sign up now

Revolutionizing how scientists
search and build protocols!