Longamp pcr buffer

Genomic DNA was isolated from each well of a confluent 24- or 96-well plate as follows: cells were incubated for 2 h at 55°C in 20 or 40 μl of lysis buffer (0.45% NP40, 0.45% Tween20, 1× NEB LongAmp PCR buffer) and subsequently heated to 95°C (10 min). One to two microlitres of this lysate was used in a 10 μl PCR reaction. PCR reactions comprised 0.2 μl DMSO (100% v/v, Sigma), 0.3 μl dNTPs (10 mM, Thermo Fisher Scientific), 2.0 μl LongAMP buffer (5× NEB), 0.4 μl LongAMP Taq (NEB) and 12 pmol of each primer. Thermal cycling was performed using the following conditions: 1 cycle 94°C for 3 min; 40 cycles 94°C for 15 s, 60°C for 30 s, 65°C for 2 min; followed by final extension at 65°C for 10 min.
For each targeted locus, two sets of genotyping primers spanning the junction of genomic sequences and targeting vector were used (left and right arms). Gene-specific primers were designed outside the 5′ and 3′ homology arms and were used in combination with primers in the knock-in cassette (either CAG-LUC-2A-GFP-IRES-BSD for targeting Rosa26 and AAVS1 loci, or Ef1α-Puromycin for the knockout experiments). To identify NHEJ-based indel formation on the second, non-targeted alleles, the region flanking the sgRNA target sites (500-600 bp) was amplified using PCR with gene-specific primers and directly assessed by Sanger sequencing. Sequences of all primers are provided (Table S2).

LR-PCR was carried out in 25μl reactions containing: approx 20ng genomic DNA, 1x LongAmp PCR buffer (New England Biolabs, M0323G), 300 μM dNTP, 0.4 μM of each primer, 2.5 units of LongAmp Taq DNA polymerase, Nuclease-free water to 25 μl. PCR amplification involved an initial denaturing step of 94°C for 30 secs, then 94°C for 10 secs, 50°C for 30 secs, 65°C for 9 minutes for 45 cycles, and a final extension period of 65°C for 10 mins followed by a 4°C hold. All reactions were carried out in a MJ Research PTC100 thermocycler.