Genotyping PCR was performed using genomic DNA extracted from the toe clipping with the primers listed in Supplemental Materials . For Sanger sequencing, PCR products were first gel-purified and cloned into the pCRII Topo vector (Thermo Fisher Scientific K460001) and sequenced with T7 or SP6 primers. Long-range PCR was performed using LongAmp Hot Start Taq 2X Master Mix (NEB M0533S) to examine genomic DNA that was purified by a Monarch Genomic DNA Purification kit (NEB T3010). The large amplicons were gel-purified and cloned into the pCR-XL-2-TOPO vector (Thermo Fisher Scientific K8050-10). The top off-targeting sites were predicted by Cas-OFFinder (Bae et al. 2014 (link)).
Protocol detail
Genotyping and Sequencing Protocol for Genomic DNA
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pCRII Topo vector
LongAmp Hot Start Taq 2X Master Mix
Monarch Genomic DNA Purification kit
pCR-XL-2-TOPO vector
Genomic
Primers
Topo
Vector
Corresponding Organization :
Other organizations : University of Georgia
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Variable analysis
independent variables
- Primer used for Genotyping PCR
- Genomic DNA extraction method (toe clipping)
- PCR amplification method (LongAmp Hot Start Taq 2X Master Mix)
- DNA purification method (Monarch Genomic DNA Purification kit)
- Cloning vector (pCRII Topo, pCR-XL-2-TOPO)
- Sequencing primers (T7, SP6)
- Genotyping PCR results
- Sanger sequencing results
- Long-range PCR amplicon size
- Predicted off-targeting sites by Cas-OFFinder
- Not explicitly mentioned
- Positive control: Not mentioned
- Negative control: Not mentioned
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