TALEN DNA constructs targeting the human CEBPB ORF were constructed using the Golden Gate Talen assembly kit (Addgene. Cambridge, MA USA).30 (link) Targeting sequences were designed using the Cornell University TAL Effector Nucleotide Targeter 2.0 web based software. Golden Gate assembly of the repeat-variable di-residue sequence was performed according to the manufacturer's instructions and the completed TALEN pairs were ligated into the pTAL3 vector. The complete TALEN ORF including the repeat-variable and FokI domains was excised using XhoI and ApaI restriction endonuceases and ligated into the pcDNA3.1(+) vector. LNCaP cells cotransfected with TALEN expression vectors targeting CEBPB were seeded in 96 well dishes, and individual clones were screened for C/EBPβ expression by Western blotting.
Golden gate talen assembly kit
Manufactured by Addgene
Sourced in United States
The Golden Gate Talen assembly kit is a laboratory tool designed for the construction of custom TALEN (Transcription Activator-Like Effector Nuclease) proteins. It provides a standardized and efficient method for assembling TALEN expression vectors from individual components. The kit includes the necessary reagents and protocols for the assembly process.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using golden gate talen assembly kit
1
TALEN-mediated Knockout of CEBPB in LNCaP Cells
2
TALEN-mediated Knockout of CEBPB in LNCaP Cells
3
CCR5 Gene Targeting Using TALEN
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To target the beginning of the CCR5 gene, the TALEN strategy proposed by Miller et al. (2011) (link) was
assembled through the Golden Gate TALEN assembly kit (AddGene, Cambridge, MA,
USA) (Cermak et al.,
2011b ). For gene editing, both right and left assembled TALEN
plasmids were transfected together, with the recognition site starting 154 bp
downstream from the CCR5 start codon (ATG) relative to the first standard
thymine (T) of the recognition site. To demonstrate TALEN activity within cells
(Kim et al., 2011 (link)),
a reporter plasmid containing the TALEN target was constructed using the
pRGS vector (red-green system plasmid), referred to as the
pRGS-CR reporter plasmid (pRGS to CCR5 Miller TALEN target) (Figure 1 ).
assembled through the Golden Gate TALEN assembly kit (AddGene, Cambridge, MA,
USA) (
2011b
plasmids were transfected together, with the recognition site starting 154 bp
downstream from the CCR5 start codon (ATG) relative to the first standard
thymine (T) of the recognition site. To demonstrate TALEN activity within cells
(Kim et al., 2011 (link)),
a reporter plasmid containing the TALEN target was constructed using the
pRGS vector (red-green system plasmid), referred to as the
pRGS-CR reporter plasmid (pRGS to CCR5 Miller TALEN target) (
4
TALEN-mediated Correction of FBN1 Mutation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Briefly, two TALENs were assembled using the Golden Gate TALEN assembly kit (Addgene) with the left TALEN binding to TCT GCA GAA ACC ATC AAG and the right TALEN binding to TCA CCC ATC AAT GAC AG. A targeting vector was constructed via subcloning of PCR fragments, generated through overlapping PCR using a Herculase II fusion DNA polymerase (Agilent), comprised of exons 21 and 22 of FBN1, flanked by a PGK-Neo fragment, and cloned into the backbone of the PGKdtabpA plasmid 54 (link).
MFS-iPSCs were first singularized with Accutase (Millipore EMD) and approximately 106 cells were transfected with 10, 10, and 20 μg of the left, right TALENs, and the targeting vector, respectively, using Nucleofector (Lonza) according to the manufacturer's protocol. Post-transfection, cells were transferred onto Matrigel-coated plate and selected for neomycin-resistant cells in the presence of 1 mg/ml G418 (Life Technologies). The survival cells were subjected to single cell cloning and multiple clones were expanded for verification of correction of the mutation and sustained pluripotency of the cells. Primers used for the verification are listed in TableS1 .
MFS-iPSCs were first singularized with Accutase (Millipore EMD) and approximately 106 cells were transfected with 10, 10, and 20 μg of the left, right TALENs, and the targeting vector, respectively, using Nucleofector (Lonza) according to the manufacturer's protocol. Post-transfection, cells were transferred onto Matrigel-coated plate and selected for neomycin-resistant cells in the presence of 1 mg/ml G418 (Life Technologies). The survival cells were subjected to single cell cloning and multiple clones were expanded for verification of correction of the mutation and sustained pluripotency of the cells. Primers used for the verification are listed in Table
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!