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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 = 151 bp
>chr6:40449344+40449494 151bp AGCTAGGACTTAGAATTGGT AACTAGTTGCACAGTACCTG
Sequence
Wt Sequence (deletion in Upper case):
taagaacaggaggatgggggcatctacagaatcatttttgtccacctagatgcaatgtcctcacttttgaaagctaggacttagaattggtttcacaattgggaaaactgtttggaagaaggggtggagcttaacaaaagcaccgATTCTTGTGTGGATTCAAGAATGAGTCTAGAATTCTGTATGCGTAAAATAAGAGCACCAGGTACTGTGCAACTAGTTCTCATTGAGCCACACAGTCTTTTAGCAATGGCATCTGTTGGACCACATATGAACTAGCTAGTTGATGTGAAACTTCCTTTTACTTCTTAAAGCCCTATTCTGACTTGCTGTTTGTCGTCCTCACCTAGAATTCGCTGGGTCAGCCAGAGATTTCTCTGCTAGAGAAGCTCTCCTCCCGAAGCTCTAGACATATGAGGCTCACACCTGCTTCCCTTATGGATGAGAAGACCTCTTTGTCTCTGGTCAATATCAACCCATTCACTCCGGAGACCTACAGAAAATTACTCCTTCAATCAAAGGGCAAGAGGAAGACCAGAGACTATCTGTAAGTGTACAGTTCTCTCAGTTTCTGAGAACTCCTTTGGGCAAGTGCTAGGTTGAGAGTAAGGTTAGGAGTAAGAACTCCACTTGaatgaagctgaagtacacgtccctcatccctacaacagtgtgcacacaggactctggcacctttctattctgatgtgcactgcaaggagcttttggataagagtttggcagaacaagaaatagatctgactttcagtagacagctga
This mutation is a 488 bp deletion of Chr6:40,449,418-40,449,905 (GRCm38/mm10)
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 70223 | AGC TAG GAC TTA GAA TTG GT | Common | A | |||
| 70224 | AAC TAG TTG CAC AGT ACC TG | Wild type Reverse | A | |||
| 70225 | CAC ACT GTT GTA GGG ATG AG | Mutant Reverse | A | |||
| 70226 | Fluorophore-1 | CCG ATT CTT GTG TGG ATT CA | Quencher-1 | WT Probe | ||
| 70227 | Fluorophore-2 | AGC ACC GAA TGA AGC TGA AGT | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 70223 | 0.40 uM |
| 70224 | 0.40 uM |
| 70225 | 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.