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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
>chr16:30599636+30599744 109bp AAAGGGATCCTCTCCTGTGC TTGCTCGCTTCACATCATTG
Mut = 107 bp
Wt = 109 bp
Fam = Mut
Hex = Wt
Sequence
Wt Sequence (deletions in lower case):
CTCTCGACCAGTGCCTGCAACCTGGTCCCTTTAAGAGACCAGAACTGCGTCCGGCGTCGCAGCCCTAAAGGGATCCTCTCCTGTGCTCCTCCCCTGTCcgcgcggtgaatggttcgtgccggcctatattaacccgagatcttcctcccggacggcaatgatgtgaagcgagcaagtcggctaccgcgcgcacgaccacagagggcgcgctgcagatcgccgagaaccccgaagcggagccggacacccccggagggaagcaatgaggtagcggcagtccccggagcggacgatgtgcgcgtcctgccctccgaaccccgcgctaccttggccgagacggccgagcgcgagtctccggcgtcccctcggagcgaggatgcagtgagctgcgggaccgctgtgcttggcccgccgcggccagccagacctctcggaccacacttggggcaagctgcttattcagggaccagtccagccaaggggctcgccctcttctgcacccactgggggtggggagcaccggcccctgggcgcactgctgtagtttccgcggctcctggccctggcccaagacttgtgtgtgtgtcacgttcgctgaagaagagttccagcttatttctctcaccatcccggttgctttggccgagccgcccacctttatttatttgtacgcactcatttggcctttttctaaaatattttccgctccgctctcttattttgttgcttgtttgagacttttgtcctggtcctcctcatctaggcttctctctctctctctctctctctctctctctctctctctctttcctgaatatttttttctaatatcctaccccagtccctgcggggttcccaagcactatctggcctctcctgcctccatctccctgccccggaggtcctcccagcctgagttttatttttcgagcttgcccgggccaagcgtagcctgggttggtggccctgcggggctctcaccccgcgctcggccaccgccgccccagccactcccgggcgtcctccggagatgctgccgtggaagaagcacaagttcgagctgctggccgaggcgccgccgcggcaggcgtccaagcctaagggctacgctgtgagcctgcactactccgcgctcagctccttggcgagagcctgcccggaaggcgcgctcagccgggtgggcagcatgttccgctccaagcgcaagaagctgcacatcaccagcgaggaccccacttacactgtgctctacctggggaatgccaccaccatccaggcgcgcggggacggttgcactgacctagcggtgggcaagatctggagcaagagtgaggccggccgtcaagggactaagatgaagctgaccgtgagtgcgcaaggcatccgcatggtgcatgccgaggagcgagcgctgcgacgtcccggccacctctacctgctgcaccgcgtcacctactgtgtggcagacgcgcggctacccaaagtcttcgcctgggtataccggcatgagctgaagcacaaggcggtaatgctacgctgccacgcggtgctggtgtccaagccggagaaggctcaggccatggctctgctgctttaccagacatcagccaacgctctggcagagtttaaacgtctcaagcggcgggatgatgcgcgtcaccagcagcaggagttggtgggcgcacacaccatcccactagtgccgctgcgcaagctgctcttgcacggaccttgctgctacaagccgccagtggagcgcagccgcagcgctcccaagctgggctccatcaccgaggacctgcttggggaacagcaagaggaggagctgcaggaggaagaagaggagcatctggaggactgcctggaggaggaggaagaagaggacggtgtgggggatggggatccagcagaggaagaggctgaggcgcagagggcgctggtggtggccatgcagttggaatgtgaggacttgctggacccgctggaaaatggccacgaggaggcgttgggggacggcggggtctcgctgggccccagctcggggacatcacttcctctgtcagtctgtgcctcagacatgaaggcccaactgtcacagcttattagtgacctgggcgaccttagctttggcaatgacgtgagcaccctggagaccgatttaagggtgacacgcctgctgtcgggcgagagcacaggcagcgagagctccatcgagggcggaggcctggatgccacccctgtcagccctggcaacccatcgggccctgctgacagcaccagcctagatgagccctactcgggctaagctgccagaagacttctctgtgcgcccctcccaacatacacatagtggctccccaacagtagttctattccccctaacctccaggaaagggaaaatgatgcacttgaagcccagacccgactttggagttttctggggaagctattccctcttgctagttgccccagcgctttggctcagaggtggctcaggttgtgaacagggggatggggggcagggagggcgagaagcactacaaaggtgggtgagggagagctggctgaagttggaaaaggccaagggcgggctggaggtgcccagcagccggtttcctgtttgtgaggcagctggggctagggaaggaggggttaacttcctctccagaccccctgactggaaggcctctttctgtcatcaatatctcattaacaaagtcactttcaaaacgttggaggcttcaatgccactttacctgcccccagttctttcggataggggagcttgggctgtttacctcagactcaaactcttgaaagtttgccaaatttctcaaataactcttcttgggggggggggcagtgtatgaaagtcgtgttttgtttacagttaaagaaacctaatatctaaaaaacaaaaaaggaattttcctttagaaatacacaccttcctctggccccaaagctcctactccatgatgctatgtaaaatatttttgcactgttgtgaagtatttttgactttttgttttttgtacataactgtgttctcggagctgaatgtttatatctcttttgctgtgcaaaagatacctgtaaaatgttgtacggttgtatatatggaaatgtgtataaaacattttgttatttttttgagcagccttgtttcagttctgttggcatgtcagcagcggggtgagaataaagaggaaaagctgtcagaacaggcgagagcctctggaaatgctaccccagatgattggcggggaggaggtgggggcggggttgtgcctccagttttagtgtggtcctgctgtggctggaagagagccggaggtgagaccttgccacccatagcgtgcttttggatcagaccaaCTCGGGGTGTTTTATCTGTGGCCAGCCTCTGTATATTTCATATTCCCATTGTGTGTGTGTGTGCAGTTTTGCTTGCTTGTTGCTTTTGAGACAAGAGACGCACTGTGCGGCTCTAACTGGCCAGGAACTTCCTAATACAGACCAGGCTGGCTTCAAATTGAGAGATCTGCTTGCCTCTGTTTCCCAAATGTTGGGGG
This mutation is a 3280 bp deletion beginning at Chromosome 16 position 30,599,668 bp and ending after 30,602,947 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 46436 | TTG CTC GCT TCA CAT CAT TG | Wild type Reverse | A | |||
| 48645 | AAA GGG ATC CTC TCC TGT GC | Common | A | |||
| 48646 | CAA GCA AAA CTG CAC ACA CA | Mutant Reverse | A | |||
| 48647 | Fluorophore-1 | CGC GGT GAA TGG TTC G | Quencher-1 | WT Probe | ||
| 48648 | Fluorophore-2 | CTG TCC TCG GGG TGT TTT ATC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 46436 | 0.40 uM |
| 48645 | 0.40 uM |
| 48646 | 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.