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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
>chr14:14348981+14349065 85bp CCATGACACCTGGGTTGG CAGAGGTCACACCTGGCATC
Mut= 85 bp
Wt= 85 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
GTTAGGCAAATTTATGAAGGCTAGAACATGATGTCATCTGGGTTTATGCAAATAAACTCAGATGGAACCATGATGTTATGCAAATCCATGCAGATTTATGCAAAGCCCATGATCAGGGCCTGTGGATATTTATTCAAATGAAATTCAAATTTATGCAAGTTCATACAAATTAATGCAAATTAGTGCTGGCCCTACCACTCACCCCAAgtatgggtgacccctgctgacgccgacatgcaggttgaccccaggtgaccccgcccctcacactgaccctgccctgccccctgacctctgtgaccccgcaggctgaccccgggcttgcccgctgctcattccagtcgctctccctctgccatgtgaccaacttcaccgtcttcctggggaaggacagggctgtggcaggatccatccagttccccccagatgatggtgggtgaggccccgcctccgtgacccccacgtgacccatgacacctgggttggcccccccaagaaccaactgtgacccctaaccctagctgtgaccctatgatgccaggtgtgacctctgatcccatgtgtgaccaggggtcctaccctggccccagcccatgacccttgaccccagggtgcgacccctgctcccaaATCTTACACCTGACCCCAACATGGGACCCCAACCCCGTGTGTGACCCCTGACCCTGTCCCCTCCATCATGACCGCAGTGACCCCATCCCAACCCCCACAGATGGCGACCACGAGGCCGCAGCCCAGGACCTGCGCTGCTGGGTGCATGAGGGGCAGCTGAGCTGCCAATGGGAGCGGGGTCCGAAAGCAACGGGTGACGTGCACTACC
This mutation is a 423 bp deletion beginning at Chromosome 14 position 14,348,720 bp and ending after 14,349,142 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 52639 | CCA TGA CAC CTG GGT TGG | Wild type Forward | A | |||
| 52640 | CAG AGG TCA CAC CTG GCA TC | Wild type Reverse | A | |||
| 52641 | AAT TAG TGC TGG CCC TAC CA | Mutant Forward | A | |||
| 52642 | AGG GTC AGG GGT CAC ACA | Mutant Reverse | A | |||
| 52643 | Fluorophore-1 | AAC TGT GAC CCC TAA CCC TAG C | Quencher-1 | WT Probe | ||
| 52644 | Fluorophore-2 | CAC CCC AAA TCT TAC ACC TGA C | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 52639 | 0.40 uM |
| 52640 | 0.40 uM |
| 52641 | 0.40 uM |
| 52642 | 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.