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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= 63bp
Wild Type = 124 bp
>chr14:51106133+51106256 124bp CTGTATGTTGAGCCATCTGT AATGGCCATTTGTATTTGAC
Sequence
Wt Sequence (deletion in lower case):
TTTTCTTACAAAACCCAAATTCCAGTTTTATGTAGATTTATCTTTCCTTGTTTTATACTCCAGTCAGGCAGGCCTGGGCAACTGACCACCCAGTCATGTTTGTTCTGACTTGTTCTGTTAGCTGTACGCCTGTCAATTAGTAAAGAGAACAGTGTTTGCTTTCAAGGAAGGCCATGTTGACAAACTAGCCACTCTCAGAGaaaggggtacaaaagggaagagcaggaagaagggggagatcttaagcagaaggaggggcaagagagggttctaagagtttagtttgcttttctcattacctctccagagcccagtccttaccatcctttctccttgttttcttctcaggcactttctaggtaatgatggatctacagaggactcagtccttgcttctgctcttggtgctgaccctgctggggttagggcttgtacagccctcctatggccaggatcgaatgtaccaacggttccttcgacagcatgtggaccctcaggtgacaggtggcaatgacaactactgcaacgtgatgatgcagagacggaagatgacttctgtccagtgcaaacgcttcaacaccttcatccacgaagacatctggaacattcgtggcatctgcagtaccaccaatatcctgtgcaagaacggccagatgaactgtcacgaaggtgtagtgaaggtcacggactgcagagagacagggaactccaaggcccccaactgtagatacagggcaagaaccagcactaggcgagttgtcattgcctgtgagggtgacccagaggtcccagtgcactttgacagatagatgctgtcctgcagctgttacagctgatagttgacctctgagtcagtgagaccaacaatgagtggtggccccaggctttgtttcctttgctcgtgccttatactcacatatatccaatataacccatggtgttaagcgcatcccatccaagggaagaaagaacccactgctcacagcactgatggcttggttctcccagattctatcccctggaacttatgcattacatgtattcagatagtatctcaacgattatagagagccttttctgtccaagctgtctcattccagtcctctaacaccccttgagaccaattctgccactgtatttagaagctttgagttaaaggatttgacgaccacaaaaaaccaaaaccaaaaccaaaaacaaaaacccagtggaagatctcagacaaaaatccaactggacgggctactcacaaaaggtgttacccacttcgttatatatactttctgttattccaaaatgtcatcattagggaagccactaagagtttggaaataggggctggagagatgcctctgtggttaagagcattggctgctcctctagaggacccaggttcaattcccagcgcccacacatcagctcacaactgtctgtaacccagttctaggtgatttgataccctcacacagacatacatgtagacagatcaccaatgcatataaaattaaaataaagattttggaaacaacatattgccaaaatgagaattacttggtattattttggtctgtggcttctaagattttttaaaatacatgaaaaatgtagtaaatcactaaaattcatttatccttctcctttgaaagattttcaatattttagtataataaagagctgtatgttgagccatctgtgtcgggatttgtgttctcctgttctttttcagttcctctctagtctatgaactctcatgtcccttagtgtccctatACATTCTGGTCAAATACAAATGGCCATTAGATCCATCATGTGACATCTGACTTTGTCTCCTTTACTCCACTGAAAGTGATCCCTCTCATACACAAGACCTGAAATAAGAACTCAGAAGTGGCCTTTGCTAAAAAGCACCAATTCTTTATGCAACAAATTGAGTGTGTTGCATGATTAAGC
This mutation is a 1570 bp deletion beginning at Chromosome 14 position 51,104,659 bp and ending after 51,106,228 bp (GRCm38/mm10)
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 68976 | CTG TAT GTT GAG CCA TCT GT | Wild type Forward | A | |||
| 68977 | AAT GGC CAT TTG TAT TTG AC | Common | A | |||
| 68978 | GGA AGG CCA TGT TGA CAA A | Mutant Forward | A | |||
| 68979 | Fluorophore-1 | CAT GTC CCT TAG TGT CCC TA | Quencher-1 | WT Probe | ||
| 68980 | Fluorophore-2 | CTA GCC ACT CTC AGA GAC ATT CT | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 68976 | 0.40 uM |
| 68977 | 0.40 uM |
| 68978 | 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.