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. 2014 Dec 31;9(12):e115318. doi: 10.1371/journal.pone.0115318

Figure 6. Simultaneous cloning of seven fragments by Hot Fusion in order to eliminate methylation sites.

Figure 6

(A) Distribution of Agrobacterium methylation sites (ME) on T-DNA of a vector encoding e35s driving the GUS gene. (B) Alignment of two clones (HF902-3 and HF902-6) eliminating ten Agrobacterium methylation sites on T-DNA. Four fragments (gB1, gB2, gB5 and gB7) were synthesized to readily mutate the multiple Agrobacterium methylation sites within the regions and three fragments (PCR3, PCR4 and PCR6) were amplified from the template with oligo primers containing the mutated base pairs. All seven fragments plus the vector were combined in one Hot Fusion reaction.