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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
>chr11:94303412-94303531 120bp AGTAACTAGCTCCAACCCAGTG GGAGTCTCTTCTACCCATGCTC
Mut= 131 bp
Wt= 120 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
CCAGCTATTTGAGAAACTGAGACAGTAAGATCTACGTTCAAGGCCAGTCTGTGCACTTAACGGGGAGTTTTATCACAAAACAAGGGGCTGGAAAATACAGCTCAGTGCTAAAGCACTTTGCTAACTTGTGTTCAATATATAGTACCACCAAAATAAACAGCCAGCTGATTATTAACCAACTGCAATACAGTATATAGTGCCTTTCCCtttgataggataacatctaaaaatatacttcatgtaagtttaggctttgagacggggtgatagggaatatatatatatggcttgtaatgtttaaaagtttatttgttttcactttatcaggtatgagaagagttctcgctttatgaaagttggctatgagagagattttttgcgatacttacagagtttacttgccgaggtagagcgcagaattagacgaggtcatgctcgtttggcattatctcaaaatcagcagtcttctggggtgagtatgaaatggacttagagtttgtaaagaactctgttctctgtagatgtggctgtctcatatcattttaaactcgacttccattcattactctgtcctttagaatacataaggtttgaggagcaccttccttctgttgtttgagggttagggaagcgtctccccccttccgcgtgcatgtttggtctgatggtgctgctggaggtcatgggctttgtatatgctagcacgtgctccaccactaaaccacacgttcgttctccagcctctgctccatgctaggaagaccagggcgggtggtcatcctagtaactagctccaacccagtgacttacttcacctccaccatgagaataggataagcaaaggggccaagactgtgtaaggagatTGCTTTTCTGAGGAGAGCATGGGTAGAAGAGACTCCTTTCTGTGACCTTTGAAGGCAAGCAAGGAGCTAGCTAGCCAGCAGAATCAAACGGAAGCCGCTCGGGTTGGGCTGCAGTAAAGCATAAAGACAGAAAAGGAGAGGAGGTTATTTCCCCAGACAGCTGTGACAAAGCTAGAGTGCAGGTGTAAAGATCTTAAGTATATTTCT
This mutation is a 659 bp deletion beginning at Chromosome 11 position 94,303,448 bp and ending after 94,304,106 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 47683 | AGT AAC TAG CTC CAA CCC AGT G | Wild type Forward | A | |||
| 47684 | GGA GTC TCT TCT ACC CAT GCT C | Common | A | |||
| 47685 | GCA CTT TGC TAA CTT GTG TTC A | Mutant Forward | A | |||
| 47686 | Fluorophore-1 | AAG CAA AGG GGC CAA GAC | Quencher-1 | WT Probe | ||
| 47687 | Fluorophore-2 | ACA GTA TAT AGT GCC TTT CCC TGC T | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 47683 | 0.40 uM |
| 47684 | 0.40 uM |
| 47685 | 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.