pAAV-Laconic and pAAV-δGlu6 were obtained by digesting Laconic/pcDNA3.1(-) (gift from Luis Felipe Barros; Addgene plasmid #44238) [25 (link)] and pcDNA3.1 FLII12Pglu-700uδ6 (gift from Wolf Frommer; Addgene plasmid # 17866) [26 (link)] by BamH1/HindIII (NEB) and cloned into pAAV-MCS (Cell Biolabs, Inc.) under of a CMV promoter. Clones were validated by sequencing. pcDNA-mito-AT1.03 (from H. Imamura, Tokyo, Japan) [27 (link)] was digested with XhoI/HindIII (NEB), blunted and cloned into the CMV promoter controlled pAAV-MCS. Mitochondria-targeting tags were two tandem copies of CoxVIII. pAAV9 were produced at UPV, Universitat Autonoma de Barcelona or at INSERM U1089 University of Nantes, France.
Xhoi hindiii
Manufactured by New England Biolabs
Sourced in Japan
XhoI/HindIII is a restriction enzyme that cleaves DNA at specific recognition sequences. It recognizes and cuts the palindromic DNA sequence 5'-C^TCGAG-3' (XhoI) and 5'-A^AGCTT-3' (HindIII).
Automatically generated - may contain errors
2 protocols using xhoi hindiii
1
Generating Recombinant AAV Constructs
2
Cloning and Verification of Bidirectional Promoters
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Promoter fragments from AHR, GATA3 and RORC were generated by PCR with ZymoTaq PCR Master Mix (Zymo Research) using the primers listed in the primer table. Inserts were ligated using GenBuilder Cloning Kit (Genscript) into pGL3-Basic (Promega) vector linearized with XhoI/HindIII (NEB) to generate constructs in both forward and reverse orientations. Each construct was verified by Sanger sequencing. Plasmid DNA for each verified construct was isolated using ZymoPURE II Plasmid Midiprep kit (Zymo Research). Preparation of two-color BII-tdT-inv-GFP vector for bidirectional promoter.
The BII-tdT-inv-GFP was obtained from Addgene (RRID:Addgene_133393). This vector contains both Green Fluorescent Protein and Red tdTomato genes in opposite orientations. The core promoter fragments from AHR and GATA3 were generated by PCR using the primers listed in primer table. PCR products were cloned into the two-color BII-tdT-inv-GFP vector by using the GenBuilder Cloning Kit (GenScript). The newly constructed vectors (BII-tdT-inv-GFP-AHR or BII-tdT-inv-GFP-GATA3) were verified by sequencing.
The BII-tdT-inv-GFP was obtained from Addgene (RRID:Addgene_133393). This vector contains both Green Fluorescent Protein and Red tdTomato genes in opposite orientations. The core promoter fragments from AHR and GATA3 were generated by PCR using the primers listed in primer table. PCR products were cloned into the two-color BII-tdT-inv-GFP vector by using the GenBuilder Cloning Kit (GenScript). The newly constructed vectors (BII-tdT-inv-GFP-AHR or BII-tdT-inv-GFP-GATA3) were verified by sequencing.
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
Revolutionizing how scientists
search and build protocols!