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Notes
This assay will NOT distinguish hemizygous from homozygous transgenic animals.
The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX).
To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility.
Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.
Expected Results
Transgene = ~300 bp
Internal positive control = 168 bp
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 21157 | CTC CCA ACC CCA GAG GTA GT | Internal Positive Control Forward | A | |||
| 21159 | CTA TAG GGC CTG GGT CAG TG | Internal Positive Control Reverse | A | |||
| oIMR1201 | GTG AAG GAA CCT TAC TTC TGT GGT G | Transgene Forward | A | |||
| oIMR1202 | GTC CTT GGG GTC TTC TAC CTT TCT C | Transgene Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| 21157 | 0.25 uM |
| 21159 | 0.25 uM |
| oIMR1201 | 0.50 uM |
| oIMR1202 | 0.50 uM |
| Glycerol | 6.50 % |
| Dye | 1.00 X |
| Kapa 2G HS taq polym | 0.03 U/ul |
| DNA |