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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
>chr9:44400086+44400175 90bp GCTCCGCACGATTTTGTTAG CCACCCAATCTCCATTACCA
Mut= 87 bp
Wt= 90 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence:
TGTCTGCTGCTTATCCACAATGAACCTGCTGATGTTGGACTCCGAAATCTGGACCCTGCCCCCTCCAAGGGCAAAAAGGgtaagcctctatcaagctgactcctagagcaccttcctttgcccctggctccgcacgattttgttaggactagcctttgccttgccctgtgcttgccactggatgtaggcgaaggaatggtaatggagattgggtgggagaagtaaagattctatcctgaaatcagccagcgcatcctgagccttctctgacagcacagatctctccattctgggtaaccccctaagtttaagcagtttgggtgccttcttccacctgcaccgtcctcatttcctttcatttgcatcgaggtaggggagagggaagcagggctttgagggggtgccccacatcaatcctg
Mutant Sequence:
CTGGCCCCCCTGTGGGAAGATCCTGAGGAAGGTGAGTGCATCTGATGAGCCTGAGGGGGAGGGCACCCAGTGGGCACTTGGGAGCACCTCTTCTTACACAACCCTTCCACCCCTAACCCATAGCAGGTTCCTTAGGAGTTCCCTGCCAGCTCCTGCTGCTGTTGGGATATTTTAGGAGCCTGAGatGTAGGCGAAGGAATGGTAATGGAGATTGGGTGGGAGAAGTAAAGATTCTATCCTGAAATCAGCCAGCGCATCCTGAGCCTTCTCTGACAGCACAGATCTCTCCATTCTGGGTAACCCCCTAAGTTTAAGCAGTTTGGGTGCCTTCTTCCACCTGCACCGTCCTCATTTCCTTTCATTTGCATCGAGGTAGGGGAGAGGGAAGCAGGGCTTTG
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 64037 | CCA CCC AAT CTC CAT TAC CA | Common | A | |||
| 64048 | GCT CCG CAC GAT TTT GTT AG | Wild type Forward | A | |||
| 64059 | TAG GAG TTC CCT GCC AGC TC | Mutant Reverse | A | |||
| 64060 | Fluorophore-1 | CTA GCC TTT GCC TTG CCC | Quencher-1 | WT Probe | ||
| 64061 | Fluorophore-2 | AGC CTG AGA TGT AGG CGA AGG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 64037 | 0.40 uM |
| 64048 | 0.40 uM |
| 64059 | 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.
Strains Using This Protocol
This is the only strain that uses this protocol.