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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
>chr11:95974915-95975059 145bp GTCCTGCACTGTCTGTGTTGA AGAAATCAGGGTTGCCCTTG
Mut= 145 bp
Wt= 145 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
AGCATGGTGGCCTGATGTGTCCCATGTCCACTGTCAAGAAGTTTAGAATATTTGAGCCTGGAATTCCTTCTCTGGGAAAGTGTGTGATATCCAAGCTTTACAGAATGGATTTAGATAATTCCAAATCTCTTGAGCCATTGAGAGAAGGAAACCATTTTTTTCTTGACATATTACCTCTGTAAATGGCTAATCCCACccactgggtgttgcttgtttgctaattgaactttcccagggtaagtttgcttccagtgtttttgtgtgtgtgtgtttgtgtgtgtgtgtgtgtttttgtgtgtgtgtgtgtgtgtgtgtgagggaggtcatctgtgaattgttttttgcttgtcctttgagtctgttcccagcgtggtagtctgctgcttatcctgttaacctacagacaggttggggtttgtcagcagagctctaactctggtggactattctgttgtctccacagtgaacactgaaagtgagacagctgtggtcaacgtcacctactctaaccgggagcagaccaggcagtgagtacctcagaagcggggctgggggctgttctgtgggtgggtccaggcttctgatattggttcagctagccctaaggtcccacaaaaggtcttttaggggtatgtttcagaaatcagaaaattctcagtaaaaatgatctgcaggaagggggatgactgggagaagagatggcttttgggtccctgggaacaacagagtcctgcactgtctgtgttgatggtgccatcttttggttatgagggggaaactgcctcgttcaaaacTGCATATATTTCTTTTTTTGAAATATATAGTGGCCTCTAATTTATTTGCCTTGGTAGCCAAGGGCAACCCTGATTTCTACCCCATTTTCAGAGCATTGTGATTGTGGGTGTGGGTTACTGTGCCTATATCAGGTATGATGCTGGATGGTTTAAGAGCCTGGGTGATGGGTTTCTAGGTCCTTTGAGTCCCCACCACCACCACAGG
This mutation is a 598 bp deletion beginning at Chromosome 11 position 95,974,993 bp and ending after 95,975,590 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 47340 | GTC CTG CAC TGT CTG TGT TGA | Wild type Forward | A | |||
| 47341 | AGA AAT CAG GGT TGC CCT TG | Common | A | |||
| 47342 | TTG AGC CAT TGA GAG AAG GA | Mutant Forward | A | |||
| 47343 | Fluorophore-1 | ATC TTT TGG TTA TGA GGG GGA AA | Quencher-1 | WT Probe | ||
| 47344 | Fluorophore-2 | CTG TAA ATG GCT AAT CCC ACT GC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 47340 | 0.40 uM |
| 47341 | 0.40 uM |
| 47342 | 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.