Cloning and Genetic Manipulation Protocol

Restriction enzymes, Gibson Assembly enzymes, Q5 and Longamp DNA polymerases were from New England Biolabs. Genomic DNA extraction kit (Quick-DNA 96 Plus Kit), mini plasmid prep (ZymoPURE Plasmid Miniprep), and gel extraction kit (Zymoclean Gel DNA Recovery Kit) were from Zymo Research. DNA maxiprep kit was from Qiagen (EndoFree Plasmid Maxi Kit). All plasmids were cloned with Gibson assembly (49 (link)) as described previously (25 (link)). All fly embryonic injections were performed by Rainbow Transgenic Flies. Cloning construct design, CRISPR guide design (50 (link)), and sequencing alignments with MAFFT (51 (link)) were done in the Benchling software suite. All primers, gRNA target sequences, and construct GenBank files are in Dataset S1.

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Oberhofer G., Ivy T, & Hay B.A. (2020). Gene drive and resilience through renewal with next generation Cleave and Rescue selfish genetic elements. Proceedings of the National Academy of Sciences of the United States of America, 117(16), 9013-9021.

Publication 2020

Gibson Assembly enzymes Quick-DNA 96 Plus Kit ZymoPURE Plasmid Miniprep Zymoclean Gel DNA Recovery Kit EndoFree Plasmid Maxi Kit

Crispr Dna polymerases Embryonic Enzymes Flies Genomic Plasmids Primers Restriction enzymes Sequencing alignments Transgenic

Corresponding Organization : University of Cambridge

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Variable analysis

independent variables

Restriction enzymes
Gibson Assembly enzymes
Q5 and Longamp DNA polymerases
Genomic DNA extraction kit (Quick-DNA 96 Plus Kit)
Mini plasmid prep (ZymoPURE Plasmid Miniprep)
Gel extraction kit (Zymoclean Gel DNA Recovery Kit)
DNA maxiprep kit (EndoFree Plasmid Maxi Kit)
Gibson assembly
Cloning construct design
CRISPR guide design
Sequencing alignments with MAFFT

dependent variables

Not explicitly mentioned

control variables

All plasmids were cloned with Gibson assembly (49)
All fly embryonic injections were performed by Rainbow Transgenic Flies

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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