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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
>chr2:28845674+28845766 93bp TGGATTCCCTTGTCATTCAG CCCTCCCCCACATAACACTC
Mut= 99 bp
Wt= 93 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
CCTGGCTTCCTGGGTAACCTCACTTGATGGGTCACTCCCTAACAGCAGCATTGAGTCCAGACTTGGTTTGTTGGGCTTAAAGCAGACTTCTAATTTCTGGATAAGTGTGATGTTGCTATGGTGGCAGATGTCTCTGTGACCTTGGGGAGAGAGCTAAGCTACCTTCTTACTGTCAAAGACCTGCATGTTTTCAGAGAATAAAGGCAAAAAAGACTGTTTGAAGTGTGGTTTCTACAAGCAGATGGACATTTTTCTTTTCCCTTTTGTATAATGGCATGAGTAAGTTTTCTAGTTACTGTGAAAAGGGAGAGAAAAAAAACATTGGAagcctggattcccttgtcattcagtcacaattgaccttttttgtgtgtgttagctatgtaggattgtttctcagcctgagtgttatgtgggggagggggccttggatagcggagttattatgaggttgccttatatatcatcagtttgtaaaagcaaatcctgcagatactatagggtagagggcagactcaccccccagctgaaagtactggcttagtgggctttaatttaatcataagatgctgatgtcgctccagcattttgtctttatgtcattgaatatatttgcattaaagtaatatttatttcatattctcttttagatttctacaataaatacagtcttaaaaatgtccagtatgaccaggtaagaggttgaagctatttttgattttcttagggtttctttgagtacaagctgtccatggaaggcagagcacattcgtgtcgggtgctgggaacaactcaaactgctaaggtcttgacttagcgcttagcctatgggactccctacctgcttgtatgcagactggatttccttccatttcggcactcaagctccctactgcaagacatagctggtttaggacattaggacatctgccatctctgaaaatgacttgagccaaaatacAGTATTTGGTCGTGTTAGTTCTTAACAGTAAAAATAAGGTCAACCAAAATGATGGGAGTAAATATTTGCTAGGATTTAGAGGGATACTGAAAAGGTATTCTAAATGCTTGTTGCATTGGAAACACTTGTAAAAGAGAAATTCAGAATTAGACTATGAGCAGTGAGCTGTGTAGTTGTAGTTGACTTTGCATAAGCAAAATGCAAAAATATGGAAGAATAGAGAACCACCAGCCTTTTCCATTTATTTGCTTTTGTGACAGTCTCATAGTTCTGCTGGCCTCA
This mutation is a 635 bp deletion beginning at Chromosome 2 position 28,845,670 bp and ending after 28,846,304 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 49010 | TGG ATT CCC TTG TCA TTC AG | Wild type Forward | A | |||
| 49011 | CCC TCC CCC ACA TAA CAC TC | Wild type Reverse | A | |||
| 49012 | TTC TAG TTA CTG TGA AAA GGG AGA G | Mutant Forward | A | |||
| 49013 | ACT CCC ATC ATT TTG GTT GA | Mutant Reverse | A | |||
| 49014 | Fluorophore-1 | ACA ATT GAC CTT TTT TGT GTG TGT | Quencher-1 | WT Probe | ||
| 49015 | Fluorophore-2 | CAT TGG AAG TAT TTG GTC GTG TT | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 49010 | 0.40 uM |
| 49011 | 0.40 uM |
| 49012 | 0.40 uM |
| 49013 | 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.