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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
>chr2:162933755+162933873 119bp TGCTTTGGATAAACTGGATGG GCCCTCGTTACCTGGATCTAC
Mut= 126 bp
Wt= 119 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence: (deleted region in lower case)
CGGAAGCCATGACGACAGCAGCCTTTTGCCATTAGACGCAGGGTGATGGTGAGGATTCCAAGGGTTAGACAAAACTGGTTAATCTGAACTAGGTGACTGTTACCTTGCGTGTTTTGTGGCCAAACCACCACCAAAAACCTCACACTGTGATGTAAGTACTTAGTGTAACTAGTAAACATTTTTGTAAAATGTAGAAATGCATGTAATCAGTTAAGTTTTATATTTTACAATGTTCTGTAAAATAAAACTTAGCGAGGTAAATTGAATAAAGGAGCAGTCACTCTCTAACAGattgtaggagaggtttagttggatttagtctatttgacttgcccttaatttaatttgtggcaaatcacaaatgtatcgagggtttagcagtataatagcaaagtcctactccagtaataaacgttgctatgtttgtactaactttcaaaaacatgcatgcctgtcattgcaacgcatctaattactcccttcatgtgataaaggtggtggaggtggatacagcagtcggagaacttctggcagagacaaatatggaccacctgttcgtacagagtacaggcttattgtagaaaatctgtctagtcgttgcagttggcaagacttaaaggtatgtacctggggttttaaagacagagcagttactttccttaagagttggctacgtggtcttgctttagatttctggagcttatcagtcttaactgttttaggatttcatgcggcaagcaggagaagtgacttatgcagatgctcacaaagaacgaacaaatgagggtgtgattgaatttagatcctactccgacatgaagcgtgctttggataaactggatggtacagaaataaatggcagaaatattaggcttattgaagataagccaagaacaagccataggcgctcctactctggcagtagatccaggtaacgagggctggggggaggcttgtcatttccagttgttgcctagttgtatttacggtgtttcttggttgtagtaagcagatattctagccacagATGGGTAGTTAAAACATATTCCTTTTTTTTTTTTAAGATTTATTTATATATTTATTTATTATATGTGAGTGCACTGTAGCTGTCTTCAGACACTCCAGAAGAGGGCATCAGATTTTGTAACAGATGGTTGTGAGCCACCATGTGGTTGCTGGGATTTGAACTCAGGACCTTCAGAAGAGCAGTCGGTGCTCTTAACCTCTGAGCCATCTCACCAGCCCCCTAAAACATATTCTTGAAAAGATTAAAACATCACTGTCTCTTGTGTGTAGATCACG
This mutation is a 736 bp deletion beginning at Chromosome 2 positive strand position 162,933,223 bp and ending after 162,933,958 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 38126 | TGC TTT GGA TAA ACT GGA TGG | Wild type Forward | A | |||
| 38127 | GCC CTC GTT ACC TGG ATC TAC | Wild type Reverse | A | |||
| 38128 | AGA AAT GCA TGT AAT CAG TTA AGT TTT | Mutant Forward | A | |||
| 38129 | AAA AAG GAA TAT GTT TTA ACT ACC C | Mutant Reverse | A | |||
| 38131 | Fluorophore-1 | AAA GGA GCA GTC ACT CTC TAA CAG AT | Quencher-1 | MUT Probe | ||
| 39197 | Fluorophore-2 | AGA ACA AGC CAT AGG CGC TCC TA | Quencher-2 | WT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 38126 | 0.40 uM |
| 38127 | 0.40 uM |
| 38128 | 0.40 uM |
| 38129 | 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.