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
>chr14:54262556+54262662 107bp AGAGGAGAGAAAACTGAAGAGTGT TGTTCTGGAGACCTGGGAAG
Mutant= 95 bp
Wild Type = 107 bp
Sequence
Wt sequence with deletion in lower case
TTCCTGAGATTAAAATATTGACCAAATCAAACTCGGGGAGGAAATGTTTATTTGGCTTACAGGTTACAATCCATCATGAAGTTTAGAAGCCAAGGCAGTAACCTAGAGGCAAGACCTGAAGCAGAGACCATAGAGCAATGTGGCTTCCTGGCTTGCTTCCCATGGCTTCCTCAGCATGTCTTCTTTTATCATTATTATTATTATCATCATCATTATTACCCTTTTCTTCTGAGACAGGGTCTTTCTATGTGGCCCTCGTTGTCCTGGAACTCACTGTGGAGACTAGGAGGGCTTCCTAAAATGATCTCTTTAAGAGGAGAGAAAACTGAAGAGTGTATTCTAGGCAAAATGTCTGTgctctcaggaagcattcttgatgctttacccaggaattcttcccttcccaggtctccagaacaagttcctggcccgttatgtatccctcccaaaccagaacaagatctggacggtgactgtgagcccagaacaaaaggatcgcacccctctggtgatggtacatggctttgggggcggtgtgggcctctggatcctcaacatggattcactgagtgcccgccgcacacttcatacctttgatctgcttggttttgggcgaagctcaaggccaacattcccaagggacccagaaggagctgaagatgagtttgtggcctcaatagagacatggcgggagaccatgggaatccccaccatgatcctcctggggcacagtttgggaggattcctggccacttcttactctatcaagtaccctgaaaggtaacttaagctactcttctcactcatgagctgactctgccttcctacacgttccaatatttatcccagtaatgctctcattccccagcaactggaaaactatgctacctccaagcaaatacagtgtatcttctgtttaatgtagagttaaacatcttatcctggtggatccatggggctttcccctacgaccaactgaccctagtgagatccgtgcacctccaacctgggtcaaggctgtggcatctgtcctgggacgttccaatccactggctgttcttcgagtggctgggccttggggtgagtcacctcccgatgaaggactctgtctttaagccaaggccatccaattcaccaccactcaccttggcgcacctacctatctctgtcatcctcactggttcttaaacactaggcctgcacatagcattgttccttaaaaccattcatcagaaccatgtctctgtattggaggtttcaggtgattgagatatatatatatatatatatatattcccttcatctgtatcagcagatagaaccagagaaaggatatatatatatatatttccttcatctgtatcagcagatagaaccagagaaaggatatatatattcccttcatctgtatcagcagatagaaccagagaaaggaagggactctgatgtgccacttctctgctacatgagtgaattttgtgtgacaacctggtagagactcagggcagtagccaccattggatccctttccatggctcaccagcaccacagcaagccggactcagatcctcagcttctaacataacacaccatggcagacaggtcccactgagccatgccctgctccagagacacaccgcatctccctctctggatttcttagcactaagtagctgtaagaagattctgagatcgagacaggacacactatagtctgtagtgtcagaggctctgcatgctctcacctcacgctgtccacgtgcctccctttgaggagcttttgacactgccgacataagaaacaggtccatttgactgaaaaggtggtgacccctgttttgtacggatagcttatgtctacatagaacttttcagtagtcacttgttagactggctgaaacgtaatggcacctaacagagtggaaaatgagccctgacatataaggtagcacaggtgttctgggataaagggagcaagtaagagacatgggtgccaaagggaagtaggcagccagagggctgacctcagagtccagggccctctaacagactcactaccatccttcctggtctaaccatgctcccctcccctggtcttggatctggccaggtccatgcaaggtatcagctatggtgttgggtagctgcctggcagccatggaagactgtgctagagggacagcagggcctgcggctatattcatggtatctcccccagatatcgctcacagacaaaaggcctgccaggacagaggcagtgtccagtgggcccggtcctcacattctgcctgcatgcctgccgcttcttcacgcacagtggtgaaagccggggccttcagggctgttaaggtcacctttccaagtattctgttacagcccgaaagtgagagagtgcttgggaaatggtatggtttcattgctaccttcagaaaagtaaaaataaataaataaataatcgcaaggttacagggtggggttggggtggaggagggggaatgcataactctgacttgaaaatcctctcaaccacagaccctcccagaacgtgtgagcctgtaaggaaagtgctggggctagctgacatgaaaattaccaacttccgtggggatggtagactgtcactaaactcattggtgaccaaaaaatcaaactctgacatttgaagtcatctgttgcttcccaacaggctcctgcactctctgtcatgttgactgcataaaggaactaagttcgtcaagagatgaactgggagaaggttgtttaaacataagcgggcaggagttcctcttctctccctgccaccaccactcactctgaccgctccacggttttgtttgttttgttttttggtggtggtggtgggggatgttgttgttctttctgttctctggtaggggcttaaaatagggtctcacacatccccagaccgaccttgagcttgaaacagttctcctgcgtctgcctcctgagtgctggaatcccaggcacgtaccacttgcttgcccagtatcacgttgttcttcctaccccctcccagggcctgggctggtgcagagattccgtccagacttcaagcgcaagtttgcagacttctttgaggatgacaccatctcggaatacatctaccactgcaatgcacagaatcccaggtgagggcggtgcccaggaccagtgcggggtggggctggggaggggctgaaaagtaaggctttgagctactgcttctcttccacagaagcctgcctcgggggaccccaggcaccagcaggcatgtctccagagttccctaggggtggtcaccagagctctaggagacaatagacttaattaaatatgctgtccctctctagaggaagacctcatgGCTTCCCTAGCGATCAGTAGCTGACATGATGAGAAACCCTCTGGGAAACATTTAAAAGATAGTTTATAACTGAAAAACTGATAAAAAAATTTTTTTGGTGTTTTGGGTTTTATTTTATAGGCAAATGTGGGGGGGGTGTTTTTAAGGCAAATAGAAAAGTGAAGTCTTTGCCCATTCACAACCTTCAGTTTCTAGATTTTCTTATTTTTAGTCTAATGTTTCATGCCATATTGATCCAGAAAGACTTCTCTCATGTGTCCC
A 3008 bp deletion beginning at Chromosome 14 position 54,262,600 bp and ending after 54,265,607 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 44793 | AGA GGA GAG AAA ACT GAA GAG TGT | Common | A | |||
| 44794 | TGT TCT GGA GAC CTG GGA AG | Wild type Reverse | A | |||
| 44795 | ATG TTT CCC AGA GGG TTT CTC | Mutant Reverse | A | |||
| 44796 | Fluorophore-1 | CTC AGG AAG CAT TCT TGA TGC | Quencher-1 | WT Probe | ||
| 44797 | Fluorophore-2 | TGT CTG TGC TTC CCT AGC G | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 44793 | 0.40 uM |
| 44794 | 0.40 uM |
| 44795 | 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.