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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= 116 bp
Wild Type =136 bp
>chr7:4642899-4643034 136bp AATTGAGCAGATTCACCCAT AAACCTGAGCCATACTGTC
Sequence
Wt Sequence (deletion in Upper case):
cccctgggactggagttgcaattgcttgtgagcaaccttgtaagagctatgcatcaaatccaggtcctctgcaagagcaacaagtgctgttagtcaattgtggcctctcagtgggttcttaggaagcctgaggagggctcagaaattgaatggggattttatttctctttggtttcccttcctagatgcAGGACCCCCTGAGCCTAATGCTGGATGTGGGGAATGAAGGCAGTGCTTCCACTTTCTCTCTGCACTCACAGCTACCTTGTACTCACTTCCTAGGTACCCTAGTGTAGCCTGTGAAATCCTGACCTCTGATGTGCCCCAGATCAACGATGCCCTGGGTGCAGATGAGTCCCTCCTGAACCGACTCTATGGCTTCCTGCAGAGTGGTGACAGCCTCAACCCGCTGCTTGCTAGCTTTTTCAGCAAAGTCATGGGCATTCTTATCAATCGCAAGACAGACCAGGTGCCTACTGACTCTCAACATGTGGGGGAGGAGTTGGGGGGGGGGTCAAGACCCTATGCTATGACACAGCCTGTGCCCCCAGCTTGTGTCCTTCCTGCGCAAGAAAGATGACTTTGTGGACCTGTTGCTGCGGCACATTGGTACCTCAGCCATCATGGATCTCCTGTTGCGTCTGCTGACTTGTGTGGAGCGGCCTCAGCTGCGGCAGGATGTCTTCAATGTGAGGAGCCTACTCAGGAAGTGGTCGGTGGAGTGGGATGGGCTGGCATTTGGTTTAGGAACTTACTGTTTTCTTCTGTCAGTGGCTCAACGAAGAGAAGATTGTCCAGCGGCTAATTGAGCAGATTCACCCATCAAAGGATGACAATGTGAGTGCTGGCTGACATCTCCAAACCTCCTTCAGATTATGTTCtatgttccttttatgctttaaaagatgggcaggggagctgacagtatggctcaggttttacagtgctttcttaggcttcaggaagccctggggccacataagccaggcagagtggctcacacctgtaatctcagttctcagggacttgaggcagagagtgcgaagccaagtatcctcctttg
This mutation is a 692 bp deletion of Chr7:4,642,957-4,643,648(GRCm38/mm10)
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 70209 | AAT TGA GCA GAT TCA CCC AT | Wild type Forward | A | |||
| 70210 | AAA CCT GAG CCA TAC TGT C | Common | A | |||
| 70211 | GGA GGG CTC AGA AAT TGA AT | Mutant Forward | A | |||
| 70212 | Fluorophore-1 | AAA GGA TGA CAA TGT GAG TG | Quencher-1 | WT Probe | ||
| 70213 | Fluorophore-2 | CCT TCC TAG ATG CTA TGT TCC T | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 70209 | 0.40 uM |
| 70210 | 0.40 uM |
| 70211 | 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.