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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
>chr9:106451272+106451365 94bp GGCTGTGGGCATGAATGA AACGGCAATGAATGGGAAG
Mut= 92 bp
Wt= 94 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
CTGATAGACTCCAGAGCTTCATTCTTTTCAGGACACTGTTAGAACGAACACAAACATAAAAAGAATTGTCTCATTCTCGCCATAGATAAAAAACACTGGGCTGTGGGCATGAATGAATTTCAGTCTACTACTGGGTGTTACGTGAacctgggcacagagtggaggaagggacccttcccattcattgccgttgtctctcctacgcccgactacctgcaggtcttgggcgatccaaggaagcagcagcccctgcccctattggggaaccggcccctggcagcttcctagctgctgtggtggataccttggagctgggacccccagttgtgatcagtccatcattaagtggcatgtattcactgcccttcctcgtggcccccgggtcccaactccggggctttgtaccagtggctcctatctgcacagacaaaatcaatgctgtggactacgccagtgtgaaggtaccgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgactgagatggtatctcaggagcaagtcggggtccatccctgggccttcttcatctgggagctgggtaaccaatggggtggtttctggggagaccccttttggttgacttagctgattgtttcgccaatccctgtataagactgtgtgctggggaaaccacaaaccaaggtctccatccactgcatcagcaccgcctctcctcctcaggagggcaggaagagactgaagaggtggtgaggcctgaagctaacctttagaagctgggctggtacgaccagcccccgattctgttctcctttccttcccctcagactccagctctcattgtatatggagaccaggaccccatgggctcctccagctttcaacacctgaagcagctgcccaaccacagagtgctggtcatggaaggggcagggcatccctgttacttggacaagcctgatgagtggcataaggggctgctggatttcctgcaagggctagcctgagactcagtcctgatgataggaggttacagactgcctgctctaggatctcttgctgtcttctcctggtctctggcttctgcctcacctgcaacaggtctgtctatacatctgtctgggttgctgtcatttctgtttttctcttgcagtgacagatcagatcaaggcggtaaatcaagtggaaaaagcgtcgggttcaaggggagaactctgattgtgggtaatccataagcttcctctacaggctccccgcttggttctcacttctgccttacttcactttctgatccatctctctcttcagccgctccacaggacagccattccttctgccgcccaccccccttagaccacagacagcatatgggtattgcttgtttagatccatcccacacccagacagaacctttcccttgggagcagcacaggggtcccacattcttgtgtagtttgcacaggtacatgtgcagtttcctgaaggatggagggaatctttgtaaacagagatccacttgccaggataaaaatgtggccaccgctagatttctcttacctacccgttgcaactggtcccagaccagaatggcctcctcagaaggggaggtgctcttcaaatctcccttaggggctgcatacctccccaggtaatcaagcacggatcttctcagccaagcccatttcctgccctgaggtttgggagtgggataaggagaggagaccgacttcggaggtccctgaaataaagtgatacaattagacctcacatgcttttgctgtctccggagcgccgcccctcccccttccccttgggactgccactgtgggctcctcccccaccctggcgggaccaaatggtccccctgccctttgtccactaggccagatggagggaaagggagattggattcccctccccacccacatacatccaggtgccaagagactcagctgaggggggaggtgtaggtcagtgggacctatatgagccgattactgaaagggggagaatatctgctctgagatgggggaagggcaggctcaattcccccccccccccacacaccCACCCACACACATATACACACACACACACAACCTATCCCTTTCCTAGTAAAGGAGTTAATGGCACTCTGACCCTAGCTCCAAAAGGAATTTGGCAGGAGATTGGAAGGATCACGGGACAGGTTGTTTAAGGGCCAAACGGCTCT
This mutation is a 1906 bp deletion beginning at Chromosome 9 position 106,451,319 bp and ending after 106,453,224 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 45064 | GGC TGT GGG CAT GAA TGA | Common | A | |||
| 45065 | AAC GGC AAT GAA TGG GAA G | Wild type Reverse | A | |||
| 45066 | AGG AAA GGG ATA GGT TGT GTG | Mutant Reverse | A | |||
| 45067 | Fluorophore-1 | ACC TGG GCA CAG AGT GGA | Quencher-1 | WT Probe | ||
| 45068 | Fluorophore-2 | TGT TAC GTG ACA CCC ACA CAC | 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 |
| 45064 | 0.50 uM |
| 45065 | 0.50 uM |
| 45066 | 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.