For in-depth product & services help, ask our
Technical Information Scientists
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
Mut= 93 bp
Wt= 110 bp
Sequence
Wt Sequence: TCTTCCACTTCCAGgtgaggaaatgggggtgtggttgagacagcagccatacctcagttaaagataacctcagagaaggCaccaaaatgttccgtgctgtagtaaggagg
Mutant Sequence:gctgtgcaccaatacaccaacttctgagcttcagctttctggtttcaaaagaagagcgctggCaccaaaatgttccgtgctgtagtaaggagg
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 38086 | CTT CCA CTT CCA GGT GAG GA | Wild type Forward | A | |||
| 38087 | CCT CCT TAC TAC AGC ACG GAA C | Common | A | |||
| 38088 | Fluorophore-1 | AAC CTC AGA GAA GGC ACC AA | Quencher-1 | WT Probe | ||
| 38091 | GCT GTG CAC CAA TAC ACC AA | Mutant Forward | A | |||
| 38092 | Fluorophore-2 | AAG AGC GCT GGC ACC AAA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 38086 | 0.40 uM |
| 38087 | 0.40 uM |
| 38091 | 0.40 uM |
| Wt Probe | 0.15 uM |
| Mutant Probe | 0.15 uM |
| 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.