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Notes
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
Mutant = 250 bp
Heterozygote = 250 bp and 330 bp
Wildtype = 330 bp
Heterozygote = 250 bp and 330 bp
Wildtype = 330 bp
Separated by gel electrophoresis on a 1.5% agarose gel.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| oIMR7318 | CTC TGC TGC CTC CTG GCT TCT | Common | A | |||
| oIMR7319 | CGA GGC GGA TCA CAA GCA ATA | Wild type Reverse | A | |||
| oIMR7320 | TCA ATG GGC GGG GGT CGT T | Mutant Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| oIMR7318 | 0.50 uM |
| oIMR7319 | 0.50 uM |
| oIMR7320 | 0.50 uM |
| Glycerol | 6.50 % |
| Dye | 1.00 X |
| Kapa 2G HS taq polym | 0.03 U/ul |
| DNA |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 94.0 | -- | |
| 2 | 94.0 | -- | |
| 3 | 65.0 | -- | -0.5 C per cycle decrease |
| 4 | 68.0 | -- | |
| 5 | -- | repeat steps 2-4 for 10 cycles (Touchdown) | |
| 6 | 94.0 | -- | |
| 7 | 60.0 | -- | |
| 8 | 72.0 | -- | |
| 9 | -- | repeat steps 6-8 for 28 cycles | |
| 10 | 72.0 | -- | |
| 11 | 10.0 | -- | hold |
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
JAX uses a 'touchdown' cycling protocol and therefore has not calculated the optimal annealing temperature for each set of primers.
Strains Using This Protocol
| Stock Number | Strain Name |
|---|---|
| 006071 | B6.129-Gt(ROSA)26Sortm1(CAG-EGFP)Luo/J |
| 006080 | B6.129-Gt(ROSA)26Sortm2(CAG-Dsred2/EGFP)Luo/J |
| 022367 | B6.129S4-Gt(ROSA)26Sortm1(CAG-EGFP/Rpl10a,-birA)Wtp/J |
| 022386 | STOCK Gt(ROSA)26Sortm1.1(CAG-EGFP/Rpl10a,-birA)Wtp/J |
| 012266 | STOCK Gt(ROSA)26Sortm5(ACTB-tTA)Luo/J |
| 5 strains use this protocol | |