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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
>chr9:21140676-21140794 119bp GATGCTGCAAGACGCCATC CTTGGACTCACAACCCTGCT
Mutant= 116 bp
Wild Type = 119 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletions in lower case):
TAGTTAAACCTAGTCTTCAAAAAAAACATAGACATGTGGCAGCAGGGCACCAGCGGATGGGTAGCACGTGGCTGTGTGCTCTGTAGTCAGGGCCCAGGGTGCGTCCCTGACCCAGGCCTGTCTGGTCCCCTGTGGTAGCAGGgtaggagttctttctatgactggctgctgattgtccctgcctgcaggcccgggtggaacgcatggagcagtttcagaaggagaaagaggaactggaccggggctgccgggagtgcaagcgcaaggtagccgagtgccagcgcaagctgaaggagctggaagtggctgagagcgatggccaggtggagcttgagcggctgagagctgaggcacagcaactgcgcaaggaggagcggagctgggagcagaagctggaagacatgcgcaaaaaggagaagaacatgccctggaatgtggacacgctcagcaaagatggctttagcaaggtgggacactggcaagagtgctatggagtgcggtcaggctagctgggggtggttcggtggtggaggctagacatgcctgtaaagaccccaggtccacccctgtgtccccatcccagtaaactagaaaaagaattctgacaatgagagccaggcaaagccaccgggtggggtgcgaggacaggggagggacagacagacagacagtttgagcttgggattggctagtggaggccaacgaagcccatccataccctctgatctgcccgaaggcccgcggccatgcctccgccaccttgatgcacttagcgacgccaccgcctccgatgtggacaggatgggcgcggcggccacggcccccaaaccccaggcgcacccctgccttgccccagcccacttgccctctcaccctgtcacagagcatggtcaataccaagcctgagaaggcagaggaggactcagaggaggcaagggagcagaaacacaagaccttcgttgaaaaatatgagaaacagatcaagcatttcggtaagttcctgcacaaaggctaggttaggagtcaaggccagcaaaaggcttccaatggcctctgcgacatcttcctaacatcctctcctgcaggcatgctccaccgctgggatgacagccagaaatacctgtcggacaacgtccacctggtgtgtgaggaaacggccaactacctggttatctggtgcattgacctggaggtagaggaggtaagtgtgcccttcccagctgcggggactcccagacactgcgcctgctcacgccccgcccacctctttgtcctggcagaaatgtgcactgatggagcaggtagctcaccagaccatggtgatgcagtttattctggagctggccaagagtctgaaggtcgacccccgagcctgcttccggcagtttttcaccaagatcaaggtaatgagctcctgtgtaccaccaggaaatccacagtgcagtgcgcgatgtcagatatttattagaggggcttgaggtgccacggggatggccgtacatatgtatacttgtgtgtgtgtgtatacatacatacatacatacatacatacatatatatatgtatgtatgtatatgaaacgtgtgtggatgtggatgtggacccatatgattaagagataactttgtgtggttggttctccacctttctgtgggtccctgtggtaaaacaagctgttaggcttacatggcaaagcactttcacctactgaacacctagctgaccctatttgaggatttctttctattcatttactttaaatattaaataatttatatgtaatgtaaatatattgcataaacattatataagtaaatattaattttttttgttgctgtgtgtgtgcctgagtgagtttatgtgcatcattcgcatgcaagtgtcttcagaggctagaagagggcgttgggtccctggaggtggagttgctagggtttgcaagctgtcatgtgggtaatgggtaccaaacttggttcctctgaaagactggggaacctgagagcagggcagggtagaacaatccagaactgaggtttctgtaagaccttgctggaggacacctgaagatgtgataccaagctggtgctgttgagccgagccaggcggctggccattagcaggtggtctcacaagctagtaggatgggcgctgaatgcaggagacaaaggccaaatcttgaggtagccatggtggagttcaggaaaagcttcagggagccttcacctccctaaacatctggttatgaattgcagtgagcagtacccaccctcttccacagaccgctgaccaccagtacatggagggcttcaagtatgaactggaagcctttaaggagcgagtgcggggccgcgccaagctgcgaatagagaaggccatgaaggaatatgaagaggaggagcgcaagaagaggctaggccctggtggcctggaccccgtggaggtctacgaatccctgcctgaggtgtgactccccagggcccaggagggaggagggtgatgtatacccattgctgctccctgcccccgcccctctgctcccacccatcccaagcccaggccctcaccctggcccctctgtactcaggagctgcagaagtgctttgatgtgaaggatgtacagatgctgcaagacgccatcagcaaaatggatcccactgtgagtccctagtccctgtggcagatggcagatggccagggGGTGCAACCCTGTCATCCAAGAGCAGGGTTGTGAGTCCAAGGCCA
A 2,555 bp deletion beginning at Chromosome 9 position 21,140,717 bp and ending after 21,143,271 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 49594 | CTT GGA CTC ACA ACC CTG CT | Common | A | |||
| 52542 | GAT GCT GCA AGA CGC CAT C | Wild type Forward | A | |||
| 52543 | CGT GGC TGT GTG CTC TGT AG | Mutant Forward | A | |||
| 52544 | Fluorophore-1 | CCT AGT CCC TGT GGC AGA TGG | Quencher-1 | WT Probe | ||
| 52545 | Fluorophore-2 | CTG GTC CCC TGT GGT GCA G | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 49594 | 0.40 uM |
| 52542 | 0.40 uM |
| 52543 | 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.