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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
>chr12:83315224-83315340 117bp AGTCACGCAAACGCACCA GGATCCCCTGCCAAACTAC
Mut = 111 bp
Wt = 117 bp
Fam = Mut
Hex = Wt
Sequence
Wt Sequence (deletions in lower case):
TGAGCCAGGTGAGTAGCTGCAGAATGTCAGGACAACTTACCTAATCCTCTCTGTCTCAAGTTTTCctgctgggaaagcagggctggcttgagaacaaaggctgtgtggtgcaggtggatccactgcggccctaaagtgtgtgtgcccgtgagcctagtggccagcatagttgtaacaactgtctcgtcctcaatgtctcctcaggctcgcggctctgcaggcggaaggaggaggcatgatgccgcctctcaggaagaccacgacaaaccctacgtctgcgacagtaagtatctccttgtactgtcatagcccaggctttctgtgccactttggcagtcacgcaaacgcaccagctctggtccacgccgggctttgctgtttccagctcccggtggtcacattccctcCTGCTGTGGCGAGGCTCTGGGCATAGTAGTTTGGCAGGGGATCCCACCTCTCAGACTGCGCTCCTG
This mutation is a 342 bp deletion beginning at Chromosome 12 position 83,315,268 bp and ending after 83,315,609 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 49935 | GGA TCC CCT GCC AAA CTA C | Common | A | |||
| 49936 | AGT CAC GCA AAC GCA CCA | Wild type Forward | A | |||
| 49938 | GAT GAG CCA GGT GAG TAG CTG | Mutant Forward | A | |||
| 49939 | Fluorophore-1 | CAC GCC GGG CTT TGC | Quencher-1 | WT Probe | ||
| 49941 | Fluorophore-2 | CTC AAG TTT TCC TGC TGT GGC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 49935 | 0.40 uM |
| 49936 | 0.40 uM |
| 49938 | 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.