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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
Mutant= 110 bp
Wild Type = 128 bp
chr1:55148831+55148958 128bp TCCCAAAGTTTCTACCACTCC CAACTACACCCACTACATTCCAC
Sequence
Wt Sequence (deletions in lower case):
TGTATGTAATTGTACATAGAAAACACCTACATAATTTTTATATAACTAAAACATCTTTCATGTTTAATCATACATATCAACAACTTCCTATCCTTCAGTTGATCATAATCAGAAGTCTTACCACAtatacacggttaatgtgaacacctttgtaacataactgtatacagttctgaaaactgcattacaatagattctcaatcaaaaagtacttgtttttctagagtcttggtgaaatttctaagtactatccagaaaaattaaacagatttttattgtataaaaggataaccagcttgaccaccttaaaaaataaaagaagaaaatacacaaagcctggataataaatgtgctttttgcatttttaggcaattctgccttgaactaaatggacttgctgtcaaacttcaggtagtattttctttatgttaaagctgtaattcaaagttctcatttttaaaaatgattttgtatgcttagatttttccagtgtttcctattatcctttttatattttatatagaaatattataacccaaatgaccattttttccattatcattctacatattaaaaaaataaaattatttttaacagagtgaatgccatccagatacctgtactcagatgacagcaactgaacaatggatttttctttgtgcagctcataaaactccaaaagaggtgagaattgagagcttccagtacctgctcattggcaccattactttgggcctgaagtgaggccaaacatctcagatgggagcatttagggaagcaagtctgcttacatggcatccaagaaagagagtgaagagtcaggagcccagtgccctcaaagacactgcccatggcctgcttcctccaactcggcctcatctcccaaagtttctaccactcctgagagactggccagctgggacccacatcttcaggagataagcccTTGGGGAACATTCCATATCCATATATGAACAGAAATATTGTGGAATGTAGTGGGTGTAGTTGTACGATATTATACTTAACACTGCTGAATTTGTTTAATATCTGTGGGTGTGATGTATGTGCAGTTGTGTGTACATGTGTGTACATGCACCCGTGGAGATGAGAGAAGTCCATA
This is a 821 bp deletion beginning at Chromosome 1 position 55,187,235 bp and ending after 55,188,055 bp (GRCm39/mm39).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 60215 | TCC CAA AGT TTC TAC CAC TCC | Wild type Forward | A | |||
| 60216 | CAA CTA CAC CCA CTA CAT TCC AC | Common | A | |||
| 60217 | CAA CAA CTT CCT ATC CTT CAG TTG | Mutant Forward | A | |||
| 60218 | Fluorophore-1 | AGT CTT ACC ACA TTG GGG AAC ATT C | Quencher-1 | MUT Probe | ||
| 60219 | Fluorophore-2 | AGC TGG GAC CCA CAT CTT CAG | Quencher-2 | WT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 60215 | 0.40 uM |
| 60216 | 0.40 uM |
| 60217 | 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.