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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
>chr7:81618675-81618777 103bp GCCTAACTTGCTGCCAATGT CTGGATGCCTGAGTGGAAGA
Mut = 99 bp
Wt = 103 bp
Fam = Mut
Hex = Wt
Sequence
Wt Sequence (deletions in lower case):
GATATTCATAAGAACGCTTAGCAGCATTGACCTAGCAAACACCCCTATCTGGTTCCTGTGTTTTTAATAAGTGTGCTCATCTAACTGTCCGTAGTCCCGGGATCTGTCCATCATTCCTGTTTCTGACACTCTTTCAAGATCCTCAAGGACACTGAACTCCTTAGTCTCTGTTGTTTATAATTGTGACTTCTGTCCTGGGGTATTTAAGGAGATATTCAAGTTTGATAGGGATTGAACATAGAAAATAGGAAGGTAAAGTTTGAATTAAAAAGCTCTGAGTGGTGGCCCCAGgtatcatctggtcagcaaatcacaacttaccaatgctgtgtggagtgttctgggcccttaggccctgtgactgattgtcttggtcagcctttctgctggttagtctacactgcactctggttcaatgagtgggtttgtccagactcataaactcatattttctcattatgcctaacttgctgccaatgtaaggacaagcagtaacatgtgaagataattcactttggccaggaatctgggtgtgaaaccccgtcttccactcaggcatccagcgtcaatggcacagacgacgaaaaggcctctcacgcgagcccagccgacactcacctcaagtctgagaatgacaagctgaagatcgcgctgacacagaggtgaggttgcttcagcacacgcgcgcgcgcgcacacacacacacacacacacacacacacacacacacgcacgcgcacacacacacacacacacacaccccgctccctgcgctgtgtttcctgaaagcctttcccaccgggcagggtcctgctcagagcatcacattctgcctcctggtcagaggacgagcagttggcttcatcacctgtctgtcggtgtcagtgagcgtgtatgagcctcttggtgcagGGTGCGTGTACGGGAGATATAAATGTGAGTGTGTGTGTGAGAACCTGCTGGATCTCTAATTGTGTTTTGCTTTTGAAAGAGGAAGACCTACTTATTTATCTCTGACAAGAGAGAGTCTTAAACATCTAAGAAAAGTATGTAAGAGCCCAAGAAACAGTGCTTCTCTCTGAGGAGCCCACACCCACACTCTCAGGTGTGCCCTGTACCTTTCTATTAACCACTGTGTTTCAATAAACTCCAACTCTACTTTTAAAAAAAGTCTAAGAAATTATGAAAAATAA
This mutation is a 621 bp deletion beginning at Chromosome 7 position 81,618,326 bp and ending after 81,618,946 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 48894 | GCC TAA CTT GCT GCC AAT GT | Wild type Forward | A | |||
| 48895 | CTG GAT GCC TGA GTG GAA GA | Wild type Reverse | A | |||
| 48896 | GAA TTA AAA AGC TCT GAG TGG TG | Mutant Forward | A | |||
| 48897 | CAA AAC ACA ATT AGA GAT CCA GCA G | Mutant Reverse | A | |||
| 48898 | Fluorophore-1 | CCA GGA ATC TGG GTG TGA AA | Quencher-1 | WT Probe | ||
| 48899 | Fluorophore-2 | CCC CAG GGT GCG TGT A | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 48894 | 0.40 uM |
| 48895 | 0.40 uM |
| 48896 | 0.40 uM |
| 48897 | 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.