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
>chr5:72941187+72941310 124bp TCTTGCGTGGTGAGTAGTTATGA CTTTGGTGATGAGTACAACCTAGA
Mut= 118 bp
Wt= 124 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
TTTGTAGCTCTAATCACGGAGAAAAGTAGAATTGATTTTGAGAAGTCCTCCTTTGGGTTCATTTGTGGTTGAGATTTGGGGGTTACGCTGCTTGCAGTACCATTTTCTTGCGTGGTGAGTAGTTATGACTAAGCGCACCCCATCATTCACCCCTActttatagtcctatactcagtaacaaatgtgccctttggtttcattttcttctaggttgtactcatcaccaaagaaaaaactcacaccaatgcagaaatctgtcagtccattagtttggtgcaggcaagttttggattacccaagtcctgatgtggagtgtgctaaaaagtcccttatccacaaacttgatcaaactatgtcaggtatgtctataacttaaattgtgaattcattaattttctttggcctgaaagcatatatgttgagtgtgtatatttaggcaggcAGTTCTTGCTTTATAGCCTTTCTGAATTTCACTGTATTCATCATGAAGGCTAGATTCATGTGATGACAGTTTTTAAAATCTTAGTCTTACTTTAGTGGATTATATCTATGAGATTATGAATTTTAATTTTTCTAATAATGACCTAAAACAGTTCCTAAAATGAAAATGTTTGTC
This mutation is a 287 bp deletion beginning at Chromosome 5 position 72,941,237 bp and ending after 72,941,523 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 44103 | TCT TGC GTG GTG AGT AGT TAT GA | Common | A | |||
| 44104 | CTT TGG TGA TGA GTA CAA CCT AGA | Wild type Reverse | A | |||
| 44105 | CTG TCA TCA CAT GAA TCT AGC C | Mutant Reverse | A | |||
| 44106 | Fluorophore-1 | CTC AGT AAC AAA TGT GCC CTT TG | Quencher-1 | WT Probe | ||
| 44107 | Fluorophore-2 | CAC CCC TAA GTT CTT GCT TTA TAG C | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 44103 | 0.40 uM |
| 44104 | 0.40 uM |
| 44105 | 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.