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RNAi vector construction and bacterial transformation

RT Ronald R. Tapia
CB Christopher R. Barbey
SC Saket Chandra
KF Kevin M. Folta
VW Vance M. Whitaker
SL Seonghee Lee
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Using pssRNAit75, two ~200-bp RNAi fragments of genes FaMLO10 and FaMLO20 and one combined ~400-bp fragment were synthesized (Eurofins, Des Moines, IA, USA) and cloned in pDONR221™ vector (Thermo Fisher Scientific, Waltham, MA, USA) using standard procedures (Table S7). The insert identity was confirmed by sequencing (GENEWIZ, South Plainfield, NJ, USA) of three clones for each target gene. After checking, the fragments were inserted into silencing RNAi Gateway® vector pK7GWIWG2(II)76 and the transformants were confirmed by sequencing both strands. The constructs were then inserted into Agrobacterium tumefaciens strain EHA105 and the transformed cells were tested by PCR using specific primer pairs for the presence of RNAi constructs (Table S7).

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