PCR analysis of the targeting events for HDR, NHEJ, and multiple copy integration

Genomic DNA isolation:

Genomic DNA was isolated using mouse ear or tail biopsies. The biopsy tissue was lysed overnight at 55^oC in DNA lysis buffer containing proteinase K (100 µg/ml) (Roche). 

Genomic DNA was extracted by phenol-chloroform method, precipitated by 2.5 volume of 96% ethanol (or 0.7 volume of isopropanol), and washed with 70% ethanol. 

Approximately 2 to 20 µg of genomic DNA was dissolved in TE buffer, and used for PCR analysis. 

PCR analysis of HDR, NHEJ, and multiple copy integration targeting events:

PCR analysis was performed similarly to quantitative TaqMan qPCR analysis. 

The donor DNA templates for targeting events were designed to contain an additional sequence in the vicinity of the LoxP site encoding a unique standard primer, and a sequence from the standard Universal Probe Library (UPL) (Merck), used for TaqMan qPCR. This allows fast and efficient detection of a homologous recombination (HR) event within the DNA regions flanking the template, as well as detection of multiple copy integrations.

qPCR reactions were performed using 2 µl (50–100 ng) of genomic DNA samples in a total volume of 20 µl containing 10 µl of 2X TaqMan Master Mix (Roche), 0.1 µM of specific UPL TaqMan LNA probe (if applied), and 0.1 µM of each primer. 

PCR amplification was performed as follows: enzyme activation at 95^oC for 5 min (Ramp Rate (RR) 4.4), with subsequent 35–50 cycles of PCR: 95^oC—10sec (RR 4.4); 60^oC—30 sec (RR 2.2) and final cooling cycle: 40^oC—10 sec (RR 2.2). 

Obtained PCR products were loaded onto 0.8% Agarose gels, in 1X TAE buffer, or 6% polyacrylamide (PAAG) gel, in 0.5X TBE buffer. DNA size markers were included. Samples were run according to the expected sizes and gels were photographed.

Agarose Gel-electrophoresis:

Dissolve 1.6 g Agarose in 20 ml 1X TAE buffer by heating in microwave. Cool for 15 min in 55^oC water bath and pour the gel. Run the gel at approximately 1V/cm.

Polyacrylamide (PAAG) Gel-electrophoresis:

Take 9.0 ml acrylamide 30% (19:1) and add 28.5 ml H₂0, 450 µl of 10% ammonium persulfate (APS), and 45 µl Temed; pour the gel. Run the gel at approximately 5–7 V/cm.
 

Solutions:

DNA lysis buffer: 100 mM tris-HCl (pH 8.5), 5 mM EDTA, 0.2% SDS, 200 mM NaCl
TE buffer: 10 mM Tris, pH 7.9; and 0.2 mM EDTA
Acrylamide 30% (19:1): Dissolve acrylamide 150g, and bis-acrylamide 7.9 g in 500 ml H20
1X TAE buffer: 40 mM Tris, pH 8.5; 20 mM acetic acid, 1 mM EDTA
0.5X TBE buffer: 50 mM Tris, pH 8.3; 50 mM boric acid, 1 mM EDTA

All PCR primers used are described in detail in Supplementary Materials.