Neb cutsmart buffer

Seven overlapping fragments (each approximately 1 kb in size) that spanned a 5.735 kb region (chr2 : 112,651,004-112,656,738) from –5187 bp to +548 bp of the MERTK transcription start site were cloned using healthy control human DNA (see above) as a template. Primer pairs were designed using UCSC genome browser with the human reference genome (GRCh37/hg19) and were sourced from Integrated DNA technologies (Coralville, IA, USA) (Table 1). PCR reactions were carried out using 2 U recombinant Taq DNA polymerase recombinant (5 U/μl) (Invitrogen, CA, USA), 1× PCR reaction buffer (Invitrogen), 2 mM dNTPs, 1.5 mM MgCl₂, 0.5μM forward and reverse primers and 100 ng of human DNA. PCR cycling conditions were as follows: 1 cycle of 95°C for 3 minutes; 35 cycles of 95°C for 30 seconds, annealing temperature (Individual T_a for each amplicon outlined in Table 1) for 30 seconds, 72°C for 2 minutes; followed by 1 cycle of 72°C for 10 minutes.
PCR amplicons and pGL3 enhancer vector (Promega, USA) were digested with appropriate restriction enzymes (NEB, USA) in 10× NEB cutsmart buffer (NEB). Each PCR amplicon (50 ng) was ligated into the reporter vector (50 ng) using 400 Units T4 DNA ligase (400 U/μl) (NEB). Plasmids were sequenced using Sanger sequencing (AGRF, VIC, AUS) to validate DNA sequence of cloned amplicons within pGL3 enhancer vector.

Sense and antisense oligo nucleotides (1 μM) representing deleted or inverted recombination products were annealed in 1× NEB CutSmart buffer (#B7204S, New England Biolabs) for 5 min at 95°C in heat block, followed by cooling down to room temperature after turning off the heat block. Annealed oligoes (1 μM) were then serial diluted to 1 × 10⁻⁴ nM (100%), 1 × 10⁻⁵ nM (10%), and 1 × 10⁻⁶ nM (1%). No oligo was used as the control. Quantitative PCR was carried out for amplification efficiency analysis using 200 ng of genomic DNA from RAG2-deficient cell line, 2.6 μl of serially diluted oligoes, 0.25 μl of forward and reverse primers at a stock concentration of 20 μM, 10 μl of iTaq Universal SYBR (#1725125, Bio-Rad), and up to 20 μl of water. Sequence of oligoes is listed in table S4. Recombination efficiency was calculated according to the formula relative level = 2^{(CT(Inversion, +100% oligo) − CT(target))}. Three independent experiments were carried out.