The vectors for Ter HR reporters described were constructed by conventional cloning methods using a previously described 6xTer-HR and RFP-SCR reporters10 (link),26 (link). pHIV-NAT-CD52 vectors were derived from pHIV-Zsgreen, a gift from Bryan Welm and Zena Werb (Addgene plasmid # 18121) 52 (link). Ter-containing plasmids were amplified in JJC33 (Tus–) strains of E. coli. siRNA SMARTpools were purchased from Dharmacon. All plasmids used for transfection were prepared by endotoxin-free maxiprep (QIAGEN Sciences, Maryland, MD).
Endotoxin free maxiprep
Manufactured by Qiagen
The Endotoxin-free maxiprep is a laboratory equipment product designed for the purification of high-quality plasmid DNA. It is engineered to ensure the efficient removal of endotoxins, which are important for applications where endotoxin-free DNA is required.
Automatically generated - may contain errors
Lab products found in correlation
Smartpool sirna, by Horizon Discovery (3 mentions)
Peg it, by System Biosciences (3 mentions)
Pspcas9 bb 2a puro px459 v2, by Addgene (1 mentions)
Cas9 p2a puro, by Addgene (1 mentions)
Zero blunt topo cloning vector, by Thermo Fisher Scientific (1 mentions)
Toptaq dna polymerase, by Qiagen (1 mentions)
Lentiguide, by Addgene (1 mentions)
Lenticrispr, by Addgene (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Lentivirus qpcr titer kit, by Applied Biological Materials (1 mentions)
11 protocols using endotoxin free maxiprep
1
Constructing Ter HR Reporter Plasmids
2
CRISPR-mediated Tagging of PLP1 Locus
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A guide sequence targeting the stop codon of the PLP1 locus was designed using DeskGen genome editing software (Desktop Genetics Ltd.). A guide sequence with minimal off-target and very high activity score was chosen and cloned into the BbsI restriction site of the Cas9 plasmid (Cas9-P2A-Puro modified from Addgene #62988, Li et al., 2022 (link)). pSpCas9(BB)-2A-Puro (PX459) V2.0 was a gift from Feng Zhang (Addgene plasmid # 629881 ). To clone the donor plasmid, a ∼2 kb PCR product was amplified from genomic DNA extracted from H9 ES cells and cloned into Zero Blunt TOPO cloning vector (Thermo Fisher Scientific). The sfGFP reporter DNA sequence was then introduced into the intermediate donor plasmid, precisely upstream of the PLP1 stop codon, using Gibson assembly (New England Biolabs, Li et al., 2022 (link)). Plasmids were transformed in competent Escherichia coli (C4040-10, Invitrogen) and purified using Endotoxin-free Maxi-prep (Qiagen), following manufacturers’ protocols.
3
Generating Stable Ter-Containing Plasmids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The 6xTer-HR reporters used were assembled using standard cloning methods described previously for the 6xTer-HR reporter (REF). Stable Ter-containing plasmids were generated and manipulated in JJC33 (Tus–) mutant strains of E. coli. All primers for conventional and quantitative PCR were purchased from Life Technologies. All plasmids used for mouse embryonic stem (ES) cell transfection and 293T cell transfections were prepared by endotoxin-free maxiprep (QIAGEN Sciences, Maryland, MD). siRNA SMARTpools were purchased from GE Healthcare/Dharmacon.
4
Mitochondrial Gene Therapy Using scAAV
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
sc-HSPCSB-hND1G3460A-mCherry was constructed as previously described27 (link), 29 (link). In brief, human ND1/m.3460G>A gene was fused in frame with Myc, and mitochondrial-encoded Cherry(mCherry) were cloned into scAAV backbones under the control of the mitochondrial heavy strand promoter including 3 upstream conserved sequence blocks (HSPCSB), where ND1G3460AMyc is followed by mCherry with a stop codon between two genes. mCherry cloned in the same scAAV backbone was used as a control (sc-HSPCSB-mCherry). Wild-type human ND1fused in frame with Myc was cloned into the same AAV backbone used as positive control and the treatment for the disease caused by mutant ND1 (sc-HSPCSB-hND1). The plasmids were purified using Qiagen endotoxin free maxiprep and then packaged with the VP2COX8 plus VP1, VP3 and helper plasmid PXX6 into recombinant virus: MTSAAV/sc-HSPCSB-hND1G3460A-mCherry, MTSAAV/sc-HSPCSB-mCherry, and MTSAAV/sc-HSPCSB-hND1.
5
Mitochondrial Gene Therapy Using scAAV
6
Dual-Fluorescent Mitochondrial Targeting Vector
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
scHSP-hND4FLAG + mCherry was constructed as previously described8 (link).9 (link). In brief, human ND4FLAG and mitochondrial-encoded mCherry were cloned into self-complementary (sc) AAV serotype 2 backbones under the control of the human mitochondrial heavy strand promoter (HSP), where ND4FLAG is followed by mCherry with a stop codon between two genes. Meanwhile, mCherry cloned in the same scAAV2 backbone was used as control (scHSP-mCherry). The plasmids were amplified and purified using the Qiagen endotoxin free maxiprep and then packaged with the VP2COX8GFP plus VP1, VP3 and helper plasmid PXX6 (3-fold excess) into recombinant virus: MTSAAV-hND4 and MTSAAV-mCherry.
7
Endotoxin-free Plasmid Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All plasmids used in transfections were prepared using Endotoxin-free Maxi Prep (Qiagen).
8
AAV-mediated delivery of NaVβ1 with Gad1 promoter
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The pAAV9-pGad1-NaVβ1-myc (AAV-Navβ1) vector consisted of a truncated mouse Gad1 promoter, mouse NaVβ1 gene-fused myc tag at C terminus, a woodchuck hepatitis virus posttranslational regulatory element (WPRE), and rabbit β-globin polyadenylation region. The mouse Gad1 promoter was amplified by PCR using the 5′ primer (5′-gatcGAATTCaCGAGACGCTTCACCCTACAA-3′) and 3′ primer (5′-gcatGGATCCaAGATCTGTCCGGGTGATCCGGTATTT-3′). The mouse Scn1b gene encoding NaVβ1 protein was amplified from an Scn1b encoding DNA plasmid (Origene) by PCR using Top Taq DNA polymerase (Qiagen). Those DNA fragments were flanked by inverted terminal repeats (ITRs) in an AAV-Navβ1 vector. AAV-NaVβ1 vector was amplified and purified by Endotoxin-free Maxi-prep (Qiagen) for AAV production. The AAV empty vector (AAV-EV) was ligated to a cytomegalovirus promoter, WPRE, and rabbit beta-globin poly(A) region flanked by ITRs to insert into an AAV genome. High-titer viruses for AAV-NaVβ1 and AAV-EV were produced at the Vector Core Facility at the University of Pennsylvania.
9
Mouse and Human CRISPR Library Construction
10
Constructing Ter HR Reporter Plasmids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The vectors for Ter HR reporters described were constructed by conventional cloning methods using a previously described 6xTer-HR and RFP-SCR reporters10 (link),26 (link). pHIV-NAT-CD52 vectors were derived from pHIV-Zsgreen, a gift from Bryan Welm and Zena Werb (Addgene plasmid # 18121) 52 (link). Ter-containing plasmids were amplified in JJC33 (Tus–) strains of E. coli. siRNA SMARTpools were purchased from Dharmacon. All plasmids used for transfection were prepared by endotoxin-free maxiprep (QIAGEN Sciences, Maryland, MD).
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!