For in-depth product & services help, ask our
Technical Information Scientists
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= 91 bp
Wild Type = 82 bp
>chr12:72656402+72656483 82bp CATGTGAGGCAGGACACACT CAACTTGGATGTCTTCAAGGAG
Sequence
Wt Sequence (deletion in lower case):
GCCCAGGACTAATCAGTCTCTGATGTGCACGAACAAGGAAACAAGAGAAAACAAAGAGCGAGGAAAGGGGAAGGAAAAAGAAGTCACAGACCTAGAACACGCACAGAGGAAAGGAGTGGTAAGGCGGAGTTGCGCCGTAAGGCGGGGTTGCACGGTAAGGCGGGGTTGCCGTGAGGCCGTGGTGGGGTGGGAGCAAGGTGAAAGGCGAAGCACTGAGCAGGTCACCCTAAGAGAAGAACTCCAGTCTCATCCTTAcatccccagggtctgatgcagcatgtgatacagtaccggtgctcacagcctggattgatgggttctacgcaggtgtgatgtgactccaacggtccttttattatctaaggagagataccagcttcccttgcctcagtctcctgcttaaggcccagtgccaatcagcacacccactcaaagtcttattatgccacaagcacacacacgagtcagctttctcagctgcatgtgagccaccttacccgaagagcatgcttgctggcacagtagccactggaaagggacacagaggcaatccccgcgatgctgttcacagtcacaatcttcccttgtttcctctctatcatgtgaggcaggacacacttggtcaaggatactgtccctatgtagttcagatttatcagctccttgaagacatccaagttggtttctagaactaaggaacgctggcttcttccaccgttgttgaccaaaatgtcaatctaagaatcagaaaatgggttcttaaagacagtctaagaaaacgattgcttgtattattaacctgaagagtaactgggacagtggtgtggttctagtagcagctacaaaaataaaataaaataaaataaaataaagggaaagtagatgttgcggcccacatctttaatctccgcacttgtgaggcagaggcaggtgggtctcttgagttcaaggccagcctggacttcagagtgagttccagaatggcctgtcacaaaaaaaaaaaaaaaaaaaaaaaagaaagaaagaaagaaagaaagaaagaagaaagaaaggaaggggggcggggaggggaggggaggggaaggggagggaagagaaggaaaagaaagaaagaaagaaagaaagaaagaagaaagaaaggaagaggggaggggaggggaggggaggggagtgggagaagagaagtgacaaagaaaggaaaagaaggaaggaaagaaggaaggaagaaaaataaaagaaaataaaaggaagcaaggaaaagaaaaggaagtagaattcacctgtggaatttccgtttgaagtaaccagtatcagaaaaagaaaagcatccgcagatattaacttcacccttgcttcttggggaaggggtgaaagtactacgtagctaaagccagctcccagccctgacctctctgtacatgctgcatctgttccatccctttgaaggggatccacagttggaaatctctttggcagtgaggcagagaagcaataccacccttcatgaacacaaaatgcaactcactttaccaaactcctgcagaacggctttggtggctgcctcatgggaacttgtatcggtcaagtcaaggggcagaaccagtatatctttttcttttaaattgccattttctgaaaaagatgcatgaagagtaagcaagccatcccagtttcactgaactcagcttccctgttaaatttaacctctaccccacaaaaaggtggcggtgaacccactccaggaggcactacctaagcatctctcagtgaccggggaagacaggtgacattgtgcaaggtctcagtggttaagagCATACACCACTTTTTCAGAGGACCTGAGTTTAATTCCTAGCACCCATGTCACAACCACCTATAATTCCAGCTCCAGAGACATCCAACATGGCCTCCAAGAGCACCTGCACTCACAAGTACAGACTCACACATATACACATAATTAAAAATAAATCTTTTTTTTTTTTAAGTTTATAGCATACACCAGGTGGTGGTAGTAGAGCATGCCTTTAATCCCAGCACCCAGGAAGCAGAGGCAGAGACAAAGCAGGTGGATTTCTGACTC
This mutation is a 1560 bp deletion beginning at Chromosome 12 position 72,656,058 bp and ending after 72,657,617 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 65813 | CAT GTG AGG CAG GAC ACA CT | Wild type Forward | A | |||
| 67903 | CAA CTT GGA TGT CTT CAA GGA G | Wild type Reverse | A | |||
| 67904 | CAC TGA GCA GGT CAC CCT AAG | Mutant Forward | A | |||
| 67905 | TGG GTG CTA GGA ATT AAA CTC A | Mutant Reverse | A | |||
| 67906 | Fluorophore-1 | CAA GGA TAC TGT CCC TAT GTA GTT CA | Quencher-1 | WT Probe | ||
| 67907 | Fluorophore-2 | AAC TCC AGT CTC ATC CTT ACA TAC ACC A | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 65813 | 0.40 uM |
| 67903 | 0.40 uM |
| 67904 | 0.40 uM |
| 67905 | 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.