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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
>chr1:192418839+192418966 128bp CTCATCTATCATGCTGGAGA CAGGAGTACATCAAAGACAG
Mut= 140 bp
Wt= 128 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence:
GTTCTAGAATCCAGACACTGCTTGGCTTGCCCAGGGAACTGAGGCATAAGAGTGCAGTCTGGCCTCATGGGTGCGGGCCCATGCCCAGGGTGTGGAGGGAAGGAGGCTGTGGGAATGGACCTCACATTGTCTCTTCCTTGTCTGGCCAGGTCCTGCAGATCTGCCCCAAGGACATGCGAGCCGACATTTGCGTGCACCTGAACCGCAAAGTGTTCAAAGAACACCCTGCCTTTCGATTGGCCAGCGATGGCTGCCTGCGGGCCTTGGCCATGGAGTTCCAGACAGTGCACTGTGCCCCAGGGGACCTCATCTATCATGCTGGAGAGAGTGTGGACAGCCTCTGCTTCGTGGTCTCAGGCTCCCTGGAGGTGATCCAGGATGACGAGGTGGTGGCCATCCTAGGTATGTGCTGCCTGTCTTTGATGTACTCCTGGTATTGTACTTGGGGTTGTCTTAGTCACTGGACAGCCACATTTAACCAGTGATTTCTCCTTGGGGCAGGCTC
Mutant Sequence:
gaatgttctactcccacaaggttcactctgacatactctaaagctgtcagctgaaatagcatctcctcagagacctttcccATGCCCGATTCCCTCCTAGCCCTGACTTTCAGATCTGCATCCTGAGCATTCCATTCTGCAGAACGTTCTAGAATCCAGACACTGCTTGGCTTGCCCAGGCTTGGGGCAGGCTCATTTCTCCATCCATTCATCCATCCAACAAAAGTTGAAGTTCCTTCTTTGAACCGATTGGTGTTTAAGGCCCTTGGGGTTTTCTCTTCAAGGGGCTGCAGGCTACTGAAAATGATGAGTAAACAAACCATCATTTGGAATATATCGGAAGGGAC
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 68628 | CTC ATC TAT CAT GCT GGA GA | Wild type Forward | A | |||
| 68629 | CAG GAG TAC ATC AAA GAC AG | Wild type Reverse | A | |||
| 68630 | Fluorophore-1 | AGG TGG TGG CCA TCC TAG | Quencher-1 | WT Probe | ||
| 68633 | Fluorophore-2 | CTT GGC TTG CCC AGG CTT G | Quencher-2 | MUT Probe | ||
| 73162 | ATT CCA TTC TGC AGA ACG TT | Mutant Forward | A | |||
| 73163 | CAA GGG CCT TAA ACA CCA | Mutant Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 68628 | 0.40 uM |
| 68629 | 0.40 uM |
| 73162 | 0.40 uM |
| 73163 | 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.