In vitro TALEN Cleavage Assay

In vitro TALEN cleavage assays were performed as previously described with slight modifications to the procedure^{14 (link)}. Briefly, 1 μg of each TALEN-encoding plasmid (pJG) was added individually to 20 μL of methionine-supplemented T7-TnT Coupled Transcription/Translation System (Promega) lysate and incubated for 1.5 h at 30 °C. Determination of protein concentrations and preparation of linear DNA for TALEN cleavage was performed as previously reported^{14 (link)}. Each reaction consisted of 50 ng of amplified DNA, 12 μL NEB Buffer 3, 3 μL of each in vitro transcribed/translated TALEN left and right monomers (corresponding to ~15 nM final TALEN concentration), and 6 μL of empty lysate brought up to a final volume of 120 μL in distilled water. The digestion reaction was allowed to proceed for 30 min at 37 °C (or 1 h where indicated), and then incubated with 1 μg/uL RNase A (Qiagen) for 2 minutes prior to being purified using a Minielute column (Qiagen). Reactions were subsequently run in a 5% TBE Criterion PAGE gel (Bio-rad), and stained with 1X SYBR Gold (Invitrogen) for 10 minutes. Gels were imaged using a Syngene G:BOX Chemi XRQ, and densitometry was performed using GelEval 1.37 software.