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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
Mutant= 115 bp
Wild Type = 137 bp
>chr9:44715014-44715150 137bp GAGAAACCGCCTTTGAAATC AAACGAAAACACCGAAGAGC
Sequence
Wt Sequence (deleted region in lower case):
TTGCTCTTCCTAAAAGAGAGGCACAGAAAGTTGGGAAAACCAAGGCAAGGAGGACCATCTTGCTGGAGCGAGTTAGAAATGACTAGACCTAGAAGTACGTACCTGGTACTGTTGCTGACTCTGTCCTGAGTCATTCAGAGTTAGAGCCAGGGAGGCCTTGCCTACCATTTCCCTTTATAGAAGGGGAACCCAGGGCAAGAGAGTTTGAATAAAgctgtagaacaggtaggtcagtggcagagtgggttgaactcagctctcctggctccttctccagagccctcgaggccccgttgccctaggatgaggtaggatggtactttcttgcttgtgcatgtatatggctccctggagccaaaaactggagcttgaggcctctttgcgtttctccctcagctgaatcggagagcctggtcaatgggaaccacacagcacaacctgcaacccgggcaccgtcagcctgtgccagccacagttccctggtgagctctattgaaaaggacctgcaggaaatcatggattcattggtgctagaggagcctggagctgcaggcaagaagcctgctgccacatccccattgtcgccaatggctaacggcgggcgctacctgttgtcccctccaaccagccccggtgccatgtctgtgggctccagctatgagaacacctctccagccttctctccactttcctcaccagccagcagcggaagctgtgctagccattcaccaagtgggcaggagccaggaccctctgtgcccccattggtgcctgcccgctcctctagctaccatttggctctgcaacctccacagtctcgcccaagtggttcccgctcctctgacagtccccggctgggcagaaaagggggtcatgagagacctcccagccctggtctccggggtcttctgacggacagtcctgccgctaccgtcttggctgaggcccgcagaaccactgagagtccccggctaggtgggcagttacctgtggtggctatcagcctgagtgaatacccatcttccggtgcccgcagccaacccgccagtattcctggcagccccaagttccagtctccagtgcctgctccccgaaacaagatcggcacactccaggaccgccctcccagtcctttccgtgagcccccaggcacagagcgggtactaacaagcagcccctctcgacaactggtgggcagaacattttcggatgggttagcagcaactcgtaccctccagccgcctgagagtccccgcctgggccggaggggcctggacagtatgcgggaactgcccccgttgagtccatctctgtctcgaagggctctgtccccactgccagcccggactgccccagatcccaagctaagcagggaagtggccgagagtccccggccccggcgctgggcagcccacgggacttcacctgaggacttctccctgactctaggagcacggggtcgcaggactcggagtccctcgcccaccctcggggagtccctggcacctcgcaagggtagcttcagtggcaggctgagcccagcttacagtctgggctctcttactggggcttcacctcgtcagagcccccgtgctcagcggaagctctccagtggggacttgcgggtacccattccaagggagaggaaaaacagcatcacggagatcagcgacaatgaggacgaacttctggagtaccaccgtcggcagcgccaagagcggctccgggagcaggagatggagaggctggtgagccaatgccagggaggctgccgctgcccaaggcggtgcctctgccagggtgtgggcaggccagccggcagaacaggaggggccatgtacagggcctgggcggattcaggatgaaccatcaggctaagtgacctgtgtctcaccatcctgataccctggagagaaaccgcctttgaaatcactaaggctttctaaattttttagttttgtatacgtgtgtgcaggtgtgtgtctgtgtgtgtagGCCAGAGATGAGTGGGTAGTGTCTTCCTCAATTGCTCTTCGGTGTTTTCGTTTGTTTGTTTTGTTTTTTTGGGGGGGGGGAGCGTTAAGACAGGGTTTCTCTGTGTAGCCTTGGCTGTCCTGGAACTCATTCTGTAGACCAGGCAGGCTGGC
1782 bp deletion beginning at Chromosome 9 negative strand position 44,716,848 bp and ending after 44,715,067 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 37251 | GAG GCC TTG CCT ACC ATT TC | Mutant Forward | A | |||
| 37252 | AAA CGA AAA CAC CGA AGA GC | Common | A | |||
| 37253 | GAG AAA CCG CCT TTG AAA TC | Wild type Forward | A | |||
| 37254 | Fluorophore-1 | CCA GGG CAA GAG AGT TTG AA | Quencher-1 | MUT Probe | ||
| 37255 | Fluorophore-2 | TTT GTA TAC GTG TGT GCA GGT GT | Quencher-2 | WT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 37251 | 0.40 uM |
| 37252 | 0.40 uM |
| 37253 | 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.