Table 1: Examples of microinjection mixes.
The plasmids and concentrations for three example microinjection mixes: one for a gpa-3::GFP reporter construct10, one for CRISPR/Cas9-mediated disruption of the Ss-tax-4 locus14,15, and one for piggyBac-mediated integration of an Ss-gpa-3::GFP construct13,17,18. strCas9 denotes the Strongyloides codon-optimized Cas9 gene. The final concentrations listed are commonly used in Strongyloides microinjection mixes.
| Microinjection mix: reporter construct | |||
| Component | Stock Concentration | Amount | Final Concentration |
| pMLC30 gpa-3::gfp | 300 ng/μL | 1.7 μL | 50 ng/μL |
| BU | na | 8.3 μL | na |
| total | 10 μL | 50 ng/μL | |
| Microinjection mix: CRISPR/Cas9 mutagenesis | |||
| Component | Stock Concentration | Amount | Final Concentration |
| pMLC47 tax-4 sgRNA | 300 ng/μL | 2.7 μL | 80 ng/μL |
| pEY11 Ss-tax-4 HDR plasmid | 400 ng/μL | 2.0 μL | 80 ng/μL |
| pPV540 strCas9 plasmid | 350 ng/μL | 1.1 μL | 40 ng/μL |
| BU | na | 4.2 μL | na |
| total | 10 μL | 200 ng/μL | |
| Microinjection mix: piggyBac integration | |||
| Component | Stock Concentration | Amount | Final Concentration |
| pMLC30 gpa3::gfp | 300 ng/μL | 2.0 μL | 60 ng/μL |
| pPV402 transposase plasmid | 450 ng/μL | 0.9 μL | 40 ng/μL |
| BU | na | 7.1 μL | na |
| total | 10 μL | 100 ng/μL | |