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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
>chr9:53390818+53390951 134bp TTCAGGGCTCAGAAATGTGT CTCGTATTCTAGCTTATTCTGAAGC
Mut= 136 bp
Wt= 134 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
TTTTACAGGGAGAAAGTTGAATTGCCACAGGGCCATCATCTTTAGAATGTGGACTGTTACTGCTTGAATGTTATTCAGAGGAGAAAAGACCAAGTAGGAGAAATGTTACTTTCTTCAATGAATTAATTTTCTCATCCACTGTTATTGAATGACTGATGAgtcccatgttaagtcagatccttgccttaaggatggaccacacatagttgctgtctgcaaggatcttccaggctgaaatgagcatggtttaccatgaacactgcaggacaaaggactttcactgagacataatgatcaggaaatggcaacgttatttctatttcgtttgcttacaggtcaaacacagttcaaagtagtggtaaaatccctttcacctaaagagctggtccgtatttatgtccctaaacctttggacagaaatgatggaacgtttttggtgagatacaggatgcacgagacggcccataaagggctcaagatagagatccttcatggcagtgaacatgtggctcactctccctatattttaaaaggtacgtgttactactcacctgaccctaggcatatatgggatacttgcttttcagaagggctgtaatgaccctgcagtcaaatgtgacagagtaaacattgtatatgtacttctggaagttcaaattttacttcattttttggcatttaaaaaatgatcattttctgagtttggagagcccttgctgctcttaccgagggcctgggttcagttctcagcaccacatggtggctcacaagtcagctgtaactccagttctaggggatccaatgtccctttctgaccagacaggtatacatgtgacatgcacatacatgcatgcagacaaaacacacacataaaacaaagaaacctcttgaaaaatcctttcctgtttcattttgtatagttcctattgctgtatctctaagtttgttcatcttttgtcctgcactgccagagttgccattaatcccctccagtgtaactttttaattacactggtaacatttttaattttcctaagctttctctctgggatgtggcagagttaattggtcataatctgaccttttcctgttatatttttacagtgtgttagatgaaattgagtcatgtttaatctggggctaattactctcagcaactgaggtgcacacaccccaagtcccaagaatgaggaggctttccagccaggctgacacaagcacttggcccagccctgcaggagcgctatgtactggacacaagcacttgacccagccttgcaggagcgctatgcactggccaccagcgttgctctgatggtcttccctgcactgggtggattccactcacagatgccctggttacaggctgggctggactctccttctcagagtcctgaaggcttttaggctttcttgtgtttagttctcacccctcatacactctgttcccaggctcagctgcctgggtctctggcatgctcagctcttccttaacacagtgagactgctcagtcctgcacacactctcttcctgagctacagtaagcagcaagctgtgtgacctgtgaggtttcccttgctagtgtcacctccaagacccactgtccttccctctctcgtgtccataatctagaaactgtgagtccacatgacaggtttgaagtaaaagtcagcgcctcttcccagtctgtgtctgaagtagaaaactccagcaaaatgaggcctaacgtcatctcagcactggggaagtagagacaggaggatctctgtgtgagcaagcttaagactagcctgggctatataagactctgtttccaaagatcacaaataaaccatcaagaaaatatattcagttaaatacatgtgctgaaattatgttagatttgtatatattgtttctaaatgtgtcttcagaaaagtcttcagaaaagttcagctgtccacttacactgatagacccctctgggttactctttcctctgagatgtgctgaccaagggggcattagggaccttgagggaaaggccctccattttctcaaaaacttaactatttctgcatttgaatgtctgaaaatcaagaatttttctgtgctaaaatggagtcactttgcatttattacttatacctcagcagaagtggaggagtcatccacgtgactggagtgtgctagtttggaagtgagcatgtgctctgtagtctgtccagggggcctagttgggggcagaggtggggctctcacatgatcactctactacgaattgacctaagataggttcacctctgtcaggagtttccagatctgccaagctgccatccggtgctcacttcttcataggtcatggtgtttgttaactcagggtgacactgtgtgtgtgttccctccttaggacccgtgtaccacgagtactgtgactgcccggaagatgaccctcaggtctggcaggaaaccctgtcttgtccagccagtgaaccacagatcgagcaggattttgtttccttccccagcattaatctccagcagatgctgaaggaggtccccacaagatttggggacgagcggggtgctgttgttcattacacaattctcaataaccacatctaccggagatctttggggaaatacacagacttcaaaatgttctctgatgaaattttgctgtcactggcaagaaaggtagggaaacaaatgaactgcttccagaaatgatatcattgtgttgttgtggcaacagttacatacttaggacgataaagactggtgagaacagatttcagggctcagaaatgtgttccagaacgagcgtgagtctcctacctttgcagatggctttgctgtaagatttaggttgaGGTGGAATCCCAGAACTCAGGTTCATAAGGCTTCAGAATAAGCTAGAATACGAGAGTGAGGCGGAACGCCAGGACAGAGCACAGGTCTTGTTTAGAAGTCTAAAGGTTCTGGAGAGCTCAGTCAGGAGATTAATGTGGGGGGTAGAGATTTGGGCAGCT
This mutation is a 2694 bp deletion beginning at Chromosome 9 position 53,388,204 bp and ending after 53,390,897 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 45410 | TTC AGG GCT CAG AAA TGT GT | Wild type Forward | A | |||
| 45411 | CTC GTA TTC TAG CTT ATT CTG AAG C | Common | A | |||
| 45412 | GAG GAG AAA AGA CCA AGT AGG AGA | Mutant Forward | A | |||
| 45413 | Fluorophore-1 | CTC CTA CCT TTG CAG ATG GCT | Quencher-1 | WT Probe | ||
| 45414 | Fluorophore-2 | CAC TGT TAT TGA ATG ACT GAT GAG GT | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| 45410 | 0.50 uM |
| 45411 | 0.50 uM |
| 45412 | 0.50 uM |
| Glycerol | 6.50 % |
| Dye | 1.00 X |
| Kapa 2G HS taq polym | 0.03 U/ul |
| 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.