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. 2016 Nov 28;6:36986. doi: 10.1038/srep36986

Figure 2. Testing of sgRNA activity and tag integration in injected embryos.

Figure 2

(a,f) Graphic representation of the tcf21 (a) and tbx18 (f) target loci and different PCR primers relevant for (b–e). (b,g) Testing CRISPR/Cas9 activity by loss of the BsrGI (b) and BstNI (g) restriction enzyme sites. PCR fragments generated using flanking primers and DNA prepared from embryos injected with appropriate sgRNA and V5-coding oligonucleotide (+) and embryos injected with control sgRNA and V5-coding oligonucleotide (−) were separated by agarose gel electrophoresis either undigested (lanes 1, 2) or after digestion with the appropriate restriction enzyme (lanes 3, 4). On both sides, ThermoFisher Scientific GeneRuler DNA ladder. (c,h) Testing for epitope tag integration. PCR was performed using flanking genomic and V5-specific primers. The tag-specific PCR fragments (red dashed boxes) were cut out from the gel and sequenced. (d,e,i,j) Sequencing of tag-specific PCR fragments.