ClaI NEB#R0197

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	Equal amounts of DNA isolated from NR-KO, iEV, and WT lines were used for E. coli transformations. For enzyme treatment of the DNA extracted from NR-KO lines, 2 μg of DNA was treated with 10 units of Exonuclease V (NEB) and/or 10 units of ClaI (NEB) in a 20 μl reaction for 1 hour at 37° C. Reactions were heat inactivated at 75° C for 30 min. An equal amount of DNA (500 ng) from enzyme treated samples, mock treated, and untreated samples were used to transform E. coli (DH5α high-efficiency efficiency, NEB).

Protocol tips

Equal amounts of DNA isolated from NR-KO, iEV, and WT lines were used for E. coli transformations. For enzyme treatment of the DNA extracted from NR-KO lines, 2 μg of DNA was treated with 10 units of Exonuclease V (NEB) and/or 10 units of ClaI (NEB) in a 20 μl reaction for 1 hour at 37° C. Reactions were heat inactivated at 75° C for 30 min. An equal amount of DNA (500 ng) from enzyme treated samples, mock treated, and untreated samples were used to transform E. coli (DH5α high-efficiency efficiency, NEB).

Publication protocol

Equal amounts of DNA isolated from NR-KO, iEV, and WT lines were used for E. coli transformations. For enzyme treatment of the DNA extracted from NR-KO lines, 2 μg of DNA was treated with 10 units of Exonuclease V (NEB) and/or 10 units of ClaI (NEB) in a 20 μl reaction for 1 hour at 37° C. Reactions were heat inactivated at 75° C for 30 min. An equal amount of DNA (500 ng) from enzyme treated samples, mock treated, and untreated samples were used to transform E. coli (DH5α high-efficiency efficiency, NEB). Resulting colonies were counted and random colonies selected for plasmid isolation. Resulting plasmids and control pNOC-ARS-CRISPR-sgNR2 DNA, were digested with AgeI, NotI, and EcorI (NEB) according to manufacturer’s instructions (NEB), and separated on a 0.9% agarose gel.

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