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= 119 bp
Wild Type = 148 bp
>chr15:79540935-79541082 148bp CAGGGATATGGTTGGCATTG CTCGGGAGAAAACAACCAAC
Sequence
Wt Sequence (deletion in lower case):
GAGTTCGAGGCCAGCCTGGTCTACAGAGTAAGTTCCAGGACAGCCAGGGTTACACAGAGAAAACCTGTCTCGGGGGGGAAAAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGGGAGAGGGAGAGGGAGAGGGAGAGGGAGAGGGAGAGGGAGAGAGAGAGAGAGAGAGACAGACAGACAGACAGACAGACTGACTTCATATGTACCCttagtttactagtaaagatagtaatgtgttcactttcttacagagtatgtgatggatgtgctgatggatcagcactttacagaacccactcccattcagtgccagggatttcctttggctcttagtggcagggatatggttggcattgcacagactggctctgggaagacattggcggtaggtaccaagtatttgacatataactccttttgttgtcctagttgacttacattttctcagttcttggggggttttggttggttgttttctcccgagacagggtttctctgtgtacttctggctgttgctctgtagaccagggtagccttgagctcagaaccacctacccctgcccccttccaagtgctggattaaaggcgtgccaccatgcctgcctcacatttccttagttcttaaacttgtagttaaagcactgtgagcaagcagggcatggtgagacctacttttaatcctagcactagggaggcagaggcaggtgcactgctctctggaaccagcttggtctatggagtgagttccaggatggccagagagtgctacatagtgaaacactgtctccaaagaaaaaaacaaaaaatttcagccctggagtatggtaattgttctattttcccctttcccattagggcattgttaatgactgactagcttgtacctgttagcctaatggcctaggtttgtatgacattgttgattctcattcctccccagtatttgctgcctgcgattgttcatataaaccaccagccatacttggaaagaggagatggtccaattgtaagtgtgttaccacttgtaaTTTTTCAGAATAGCCATTTTTAATGATGGTTGCATTTTATGCACTGTGCCCAAGTTAATTCTTTTTTTATGGGTCATCTCTCCAGACCCTGCCCTTATTCAGTTCTCACCACTTTTCTCCCTATGTAGAGGTGGTACTGGCCTGTCTTATTTGGAATTAAATGAATTGCCCAAAATTGTGCAGCAAAAAAATACGAATTTGAGGCATTCTTGCTCTTTTCTGTTTGTGATCTGGTTTATGTCCTGTGTGTGCTAGACAGTAATCCCACTGCTGAGCT
This mutation is a 820 bp deletion beginning at Chromosome 15 position 79,540,391 bp and ending after 79,541,210 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 67933 | CAG GGA TAT GGT TGG CAT TG | Wild type Forward | A | |||
| 67934 | CTC GGG AGA AAA CAA CCA AC | Wild type Reverse | A | |||
| 67935 | GAC AGA CTG ACT TCA TAT GTA CCC TTT | Mutant Forward | A | |||
| 67936 | AGG GCA GGG TCT GGA GAG AT | Mutant Reverse | A | |||
| 67937 | Fluorophore-1 | AAG ACA TTG GCG GTA GGT ACC | Quencher-1 | WT Probe | ||
| 67938 | Fluorophore-2 | CAG AAT AGC CAT TTT TAA TGA TGG TTG C | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 67933 | 0.40 uM |
| 67934 | 0.40 uM |
| 67935 | 0.40 uM |
| 67936 | 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.