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= 76 bp
Wild Type = 90 bp
>chr19:10028215+10028304 90bp CTGCCAGGCTGTGGTAGC TCTGCCTGTAGCATGTCGAT
Sequence
Wt Sequence (deletions in lower case):
GGACCCATGAATACTTAGTGGGAACCCTAGGAAGTTCCCAGATGCCGCCTTCAGCAGAGTCTCACAATGTGACCTGAGGCAGGTCATGTCTCCTGTCAGCCTTGGGTGCCCTATCTCTAGAATAGATAATCTTAGAGGTTGTTCCAAGGGTAAGGAGACAGGTTAGGTACTGCTGGGGATGCTTGGCTGCCAGGCTGTGGTAGCCATGGTGACATTAACCCCATgttggggttgatggtcatcttgtctgggacagatcgacatgctacaggcagaggtgacagccttgaagacattggtcatcacatccacaccagcctctcccaaccgtgagctccacccacagctgctgagccccaccaaagctggaccccgaaagggccactcacgtcaaaagagcaccagcagctccctctgccctgttgtgtgccccactgcagggcatatccctacccctgacaaagaaggcaaggaggtgaggcacaggctgcaatagtgggctcacaatcgggagggttgatcatgtaaaccagccaagtcagcgtcctgaaaaagctatcctgacccctcctgtggaacctctttctctcctcgctgggcttcagggttccctggaaccacgaaggctgggtaggctgataccaagcaggaggtgataatctttgaacttgaggctgttaggacctgaaggtcctctctcaggacatctcttcagaggtgcattttctgtgttgagtgataggtgcactggggctacctggtgttttgtgactacagtctttttttgtttttgtttttgtttttttaagatttatttatttatttttatgtgtgaatacactgttgctgacttcagaaacaccagaagagggcatcagatcccgttacagatggttgtgagccaccatgtggttgctgagaattgaactcaggacctctggaagagcagtcagtgctcttagccgctgagccatttcactagcccgtgattacagtcttaaggtctaagaccacactgtgacacgtgctgaagcagctacggccttagacaaatgtctctctcctgcctcaacaggaaatgcagggtgagaggcttggtccaggctggactgacatgaccataggccattgggtagaatttccatcacttcacacgccacataccatgcctcccttagggtgaggataaaaagccagcctttttagtatcagagggttaccggggagacatgggtgagatttagcttcctggactcttgctcaagcccaccctagctcagaactttcttcatccgtcccctgagcttctaggaaggaagtgcaagtcctttcagtccccttctgagcaccgcgcttcccaccggacactgacatctcctccttcctcctgtctgtctctgtttgtctgtttgtctatccatctgtccacccatcccctggcccagcctgggctgccccctctactctccctgctcctgtgcgtgatccccagcaaggccatccacctcacctacgagccgacctggcagctcctatctagggagccgcccacgacccccaactccgatacctctctctccttttcccgacaggtaatggctcctttaggggccaccctctctctccccctctgggcttcactcactgtgcatgcagcccagggcaggccattgttgccccttccagtgggcagcagggaaacctgtgaaatgttctctcctgtgtgcatgtacctatatgtgtatcctggacatagagggaaacctgcgggttcctatggggacccacaggttctcacaggcttgctcttgatgtttcttccaaaaggagtcagtagcagcattcaggccaaggccttggatccaagtctgtaaaattgccgtgccaagaaacttaatgtgcacccagacacaaactgtgttgttcttaattctttggtgcaaagggcagacaggacctcttctttgcctggactctgctccctgcatggctgctgagggcaggcctgatggtccactgaggcctcctgcatctccctgctcttctctccatcagctctgctttctctggtgtgctcactctcatccctgactgcccccacttggctcccctctcctctaggtggatacgacgctgtttgcagagttccaagcttggagagcttcacctaccctggataagagctgccccttcctagaaagggtgtaccgggaggacgtgggcccttgccttgacttcacagtgcaggaggtgaggtgggctgacagtggttagctggggatccagcccagtagttccctgtcacccagcatctcaatgctcttccctgatttgtcacagctctcagctctggttcggactgctgtggaggacaacacactcaccattgagcctgtggcttcacagacactgcccgctgtgaaggtgcccactgttgagtgtaacaacaccaagtaagcttagtgtgctgggtcccttcctgcccacatgggaaagtccagcatggagccttgggtagcaccctggggGGGACTGGGATATGAGCCTGAGTCTTGGGCCTCCTAGATGGATATAGAGCCTCAGGGTGGGCAGGGACTATAACATTCAGGGTGACTGACTGTCTCCCGGGATTCATGACCTAACCTAGGGACTCCCAAGAGATGGGCATGGGTCTGGGGGATATAGTTCAGTAAAGTCCTTGACTAGCATGCACAAAACCA
This mutation is a 2304 bp deletion beginning at Chromosome 19 position 10,028,253 bp and ending after 10,030,556 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 63992 | CTG CCA GGC TGT GGT AGC | Common | A | |||
| 63993 | TCT GCC TGT AGC ATG TCG AT | Wild type Reverse | A | |||
| 63994 | TCT AGG AGG CCC AAG ACT CA | Mutant Reverse | A | |||
| 63995 | Fluorophore-1 | TTG ATG GTC ATC TTG TCT GGG AC | Quencher-1 | WT Probe | ||
| 63996 | Fluorophore-2 | ATT AAC CCC ATG GGA CTG GGA T | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 63992 | 0.40 uM |
| 63993 | 0.40 uM |
| 63994 | 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.