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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
>chr6:56723728+56723860 133bp GGTGAGACATGCACAAGAGA ATTGAAGGCACACCCATCAG
Mut= 118 bp
Wt= 133 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case; and insertions with carrots ^g^ (T insertion):
TTCAAATCCCAGCAACCACATGGTGGCTCACAACCATCCATAATGAGATCTCATGCCCTTTTCTGGAGTGTCTGAAGACAGCTACAGTGTACTTACATATGATAAATAAAAATAAACCTAAAAAAAAAGAAAGAAAAAATTGTGAGTGTTCATGTAGAAAGGAGGTTAACTGTTTTGAAATCAATTAGCAAA^ctcatctaatttttaagtacaagttccaattgtagtaattttaccagcaggaaacaagaattctaaaaactgccaacataaggtaggcagtctacaatttatggatgaattgtgtttccagagttgctatttaaagcctggaccacattttcccaaagaaactgtgttacagacaccgtgaacccaattcttcctgacacggtcttaatttcacttacagaattcttgagtatgtttagcacctaaactctatttaaacatactcaactctatttaaactttgagttacaactagaccctttaaatcagctgggaatagtctataaaccttcccatctttctccacttctgcttttgagtttgttgataatcttcaatagcatcattcagggtttaagcacatgacagttcatcagtgtaagaccctttctttgatcactgtttggctaacattctttttcattttaggttgaattctcttacccaccactaattccaggagatggacatgatagccacactttacctgaagaatggaagtacttgcctttccttgccttaccagatggtgcacataactatcaggaaggtatgtcattaaatcgtagaccaattactaagtgcaattttggaaattttctcatagcggtcgtgaatactttggataaatgaaagcctttggacctgttgtgattacttataaaatgtgtatgtgttggaggtgagacatgcacaagagaagcttcagagagtactgggcctatttccttggttagtaaccctcgg^CTCCCTCCTGCCCAGAGCTCTTAGGTATAGCCAGAGCAGGGCTGCTGCTGATGGGTGTGCCTTCAATTATATTATGCTTGAGACTGAACTCAGTGGCAGAAATGAAGATTTGCCTGCATTAGCTGTACATGGTCTGGAAAGCTCTGGTTTCAGACTTACTCAACCATCATCCTCTAATGCCCACCTGGATACAGTGG
This mutation is a 786 bp deletion beginning at Chromosome 6 position 56,723,008 bp and ending after 56,723,793 bp (GRCm38/mm10). In addition, there is a single bp (T) insertion at the deletion site.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 47753 | GGT GAG ACA TGC ACA AGA GA | Wild type Forward | A | |||
| 47754 | ATT GAA GGC ACA CCC ATC AG | Common | A | |||
| 47755 | TGA GTG TTC ATG TAG AAA GGA GGT | Mutant Forward | A | |||
| 47756 | Fluorophore-1 | ACT GGG CCT ATT TCC TTG GTT A | Quencher-1 | WT Probe | ||
| 47757 | Fluorophore-2 | CAA ATC TCC CTC CTG CCC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 47753 | 0.40 uM |
| 47754 | 0.40 uM |
| 47755 | 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.