Generating Homology-Directed Repair Templates for GFP11 Insertion

The 4× GFP11 double-stranded DNA (dsDNA) donor templates [258 base pairs (bp)] for both N- and C-terminal insertion were synthesized in the pUC57 vector by GenScript (New Jersey, USA), containing four repeats of GFP11 separated by five amino acid linkers, as shown by Leonetti et al. (54 (link)). Homology-directed repair (HDR) templates were generated by polymerase chain reaction (PCR) amplification of the 4× GFP11 donor template with gene-specific primers (IDT, Iowa, USA) using Phusion DNA polymerase (NEB, Massachusetts, USA), resulting in HDR templates with 35- to 45-nucleotide (nt) homology arms. The final PCR product was confirmed on a 1 to 2% agarose gel and purified using the GeneJET PCR Purification Kit (Thermo Fisher Scientific, Massachusetts, USA). The concentration of the HDR template was measured on the NanoDrop before cell transfection.

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Hastings J.F., Latham S.L., Kamili A., Wheatley M.S., Han J.Z., Wong-Erasmus M., Phimmachanh M., Nobis M., Pantarelli C., Cadell A.L., O’Donnell Y.E., Leong K.H., Lynn S., Geng F.S., Cui L., Yan S., Achinger-Kawecka J., Stirzaker C., Norris M.D., Haber M., Trahair T.N., Speleman F., De Preter K., Cowley M.J., Bogdanovic O., Timpson P., Cox T.R., Kolch W., Fletcher J.I., Fey D, & Croucher D.R. (2023). Memory of stochastic single-cell apoptotic signaling promotes chemoresistance in neuroblastoma. Science Advances, 9(9), eabp8314.

Publication 2023

pUC57 vector Phusion DNA polymerase GeneJET PCR Purification Kit

Agarose Amino acid Arms Cell Dna polymerase Donor Dsdna Gene Homology directed repair Nucleotide Polymerase chain reaction Primers Transfection Vector

Corresponding Organization : UNSW Sydney

Other organizations : Cancer Institute of New South Wales, Sydney Children's Hospital, Ghent University Hospital, Cancer Research Institute Ghent, Ghent University, University College Dublin

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Variable analysis

independent variables

Homology-directed repair (HDR) templates generated by polymerase chain reaction (PCR) amplification of the 4× GFP11 donor template

dependent variables

Concentration of the HDR template measured on the NanoDrop before cell transfection

control variables

4× GFP11 donor template in the pUC57 vector
Gene-specific primers (IDT, Iowa, USA) used for PCR amplification
Phusion DNA polymerase (NEB, Massachusetts, USA) used for PCR amplification
1-2% agarose gel used to confirm the final PCR product
GeneJET PCR Purification Kit (Thermo Fisher Scientific, Massachusetts, USA) used to purify the final PCR product

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