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Fig. 2 | BMC Biology

Fig. 2

From: Comparative analysis of single-stranded DNA donors to generate conditional null mouse alleles

Fig. 2

Screening strategies for HDR and NHEJ alleles, and random ODN insertion. Relative positions of the primers and approximate sizes of PCR products are listed below each allele. Scissors represent target sites. a Genotyping schemes for detecting loxP donor sequences. Orange triangles represent loxP sites, with representative homology sequence color coded on blue DNA strand. b Genotyping for NHEJ events utilizes a three-primer system, with P1 being shared between P2 and P3. Primers P1 and P3 reside between 100 to 200 bp outside of the target site (an average deletion product size is depicted). The P1 + P3 primer pair may not always amplify a wild-type product, if the target sequences are too far apart. 1400 bp represents the average distance between loxP insertion sites. c Random ODN insertion PCR primers reside internal to homology arm sequence, and will amplify the expected size product if the ssODN donor has been incorporated elsewhere in the genome, away from the critical exon, in addition to the on-target locus. d Primers for the homology arm screen were used in a SYBR-green quantitative PCR reaction from DNA samples from the N1 generation, using β-actin as a two-copy normalization control

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