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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
>chr7:16076026+16076137 112bp CGAGTGCTTTTGGTTTGGA TGGATCAGGTTGAGCTTCTAGG
Mut= 96 bp
Wt= 112 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletions in lower case):
AGTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTTTCTCTCTCTCTGTGTGTGTGTGTGAGAGAGAGACACACAGAGAGAGAGAGACAGAGAGAGAGCGAGTAAATAAAGCACTCACCTCCTCTAAGCTCTCTAATCAGATATAAGGTTGTTTCTAACATTTTACTAGAAAAAGCAGGTAGATTTTTTTTttTTATAAAAAGGTGAGAACCCAAGAGCAGAGCAAGATGGCGAGTGCTTTTGGTTTGGATAATGCATCTCTAGATAGACATTGTATGGGTAGGTTGGCTTctacccctgcttgtcttatcatactgctaccctagaagctcaacctgatccatacctgcacatcttgcaccaagacctggtgcagttctatagataaggcctggtccagacctcagctaagccttgtcgtctggccgtgcagagcctctgaggtgggggagtctcagactactgggacagggctctgtgctgagggtctatattgctttagcactcccctatcctatctcagcataacctgtgctttctccctttgtttagacctggggctgggcgcactcaaggtgcctaaagaggctgatgaaggaggcagggccactgggagtacaaggaaaggcaagcgacagcacagttcccctcagaatccactcctggactgcagcctgtgtgggaaagtgttcagcagcgccagttccctgagcaagcattacctgacgcacagccaggagagaaaacacgtgtgcaaagtctgcagcaaagccttcaagcggcaggaccacctgtatgtcaagaatctggccagaagggcagggggcatgcacctgtcctcctaccctctcccccatccatctggtcctctgttgagttcccacttctgtgcttctcagatgtatgtcccctgtcctccagtgtggactctgttctCTTCACACTGCTATTGTCTGGGGGCAGTCTCTTTACTAAGCCCCAAATTTACACCAGTGAGGAAATCCCTGTCTATTTGCAGACATGGCTCTTGTGGTTTCTAAATGTGATGAGTTACCAGGCGTGCTGGCACACTGGGGAGGTAGAAGCAGGAGGATCAAGAGTCTAAGGCCAGCCTCAACTACATACATTACAAGTTTAAAGCCCACC
This mutation is a 645 bp deletion beginning at Chromosome 7 position 16,076,086 bp and ending after 16,076,730 bp (GRCm38/mm10). In addition, there is an additional 2 bp deletion (TT) 98 bp before the 645 bp deletion site.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 48990 | GTA AAG AGA CTG CCC CCA GA | Mutant Reverse | A | |||
| 48991 | CGA GTG CTT TTG GTT TGG A | Common | A | |||
| 50047 | TGG ATC AGG TTG AGC TTC TAG G | Wild type Reverse | A | |||
| 50048 | Fluorophore-1 | CCC TGC TTG TCT TAT CAT ACT GCT A | Quencher-1 | WT Probe | ||
| 50049 | Fluorophore-2 | AGG TTG GCT TCT TCA CAC TGC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 48990 | 0.40 uM |
| 48991 | 0.40 uM |
| 50047 | 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.