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Notes
Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
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= 110 bp
Wild Type = 110 bp
Sequence
Wt Sequence:
GTGGCACTTGGCGGGGTGCTTGCTTACAGACCGGCCTCTCTGTGTCTCCACAGGTTTACAAGTCGTCCTGAAGTCAATCATGAAGGCGATGATCCCTCTGCTGCAGATCGGCCTCCTCCTGTTTTTTGCAATCCTTATTTTT
Mutant Sequence:
GCTCCATGGGTGGCACTTGGCGGGGTGCTTGCTTACAGACCGGCCTCTCTGTGTCTCCACAGGTTTACAAGTCGTCCTGAAGctgATCATGAAGGCGATGATCCCTCTGCTGCAGATCGGCCTCCTCCTGTTTTTTGCAATCCTTATTTTT
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 67170 | GGG TGC TTG CTT ACA GAC C | Forward | A | |||
| 67172 | AAC AGG AGG AGG CCG ATC T | Reverse | A | |||
| 67173 | Fluorophore-1 | CGT CCT GAA GTC AAT CAT GAA G | Quencher-1 | WT Probe | ||
| 67175 | Fluorophore-2 | CGT CCT GAA GCT GAT CAT GAA GG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTPS-kapa | 0.26 mM |
| 67170 | 0.50 uM |
| 67172 | 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 | 95.0 | -- | |
| 2 | 95.0 | -- | |
| 3 | 60.0 | -- | |
| 4 | -- | repeat steps 2-3 for 40 cycles | |
| 5 | 40.0 | -- | Forever |
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
Strains Using This Protocol
This is the only strain that uses this protocol.