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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
>chr14:31482590-31482689 100bp TAACACACAAGTCCCAAGTCC TCTACTCTGCGGGCCTCACT
Mut= 124 bp
Wt= 100 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence: (deleted region in lower case)
AACTGTTTTATGCTCCATCATATCTAAAAAAAGTGACTTCTAAGGCATTTTATTTACTTATTGGTTTTTTGAGACAGGATCCTTTTGCTAGGTAAACCAGGTAGGCTAGGTACTGTGTAGGTCCGGCTAACCTCAAACTCAAAGTGATATTCCTGCCTCCCTCTTGAACTCTGTGATTACGGTATGTACTCCACACCTGACATGGTTTTTTAAAGAGCAACCATTCATAGTAAGGGTATTTTGTATGCCTTCTTTCACCACTCCCACTggtctgtgttttaattcctaatgtcatcagattacattttagttgaccgggtttatacagcctgctttattacttttgtaggtattaaaaccaggcagaagtgtcttattccgtgactacgggctgaatgatcacgccatgcttagatttaaagctggaagcaaacttggagaaaatttttatgtcaggcaagatggaaccagatcgtatttttttactgatggtaagaggaatggagtgtctccttagagtgtgtctttctctgctctgtgtacttgactgcaccattagtggagtgctgcctgggcaggggtgtggccgtgctaacacacaagtcccaagtccttagagcaagttctgatacaccacatacccagccctggcctgtggGGACATGACTAGTGAGTGAGGCCCGCAGAGTAGACAGTCCTCAGATCTGTCATGTGATGCCAACAGGAACTGTGCTGGAAACGGGGAAGTAGGACCAGAAAGTCACATCTTCCAAACAAGTGCCTTGAGTTCACCATGGCGGCCTGGGGACACTTTTCCAGACCCACAGGCAAAACTCCTTGACTAAATTTGCTGGCACAGAGCACTGGGAATGTGGCTCAGGTGGTGTAGTGCTGACCTGGCATTGCTCAGTTTCTGGATCCAGTCCC
This mutation is a 390 bp deletion beginning at Chromosome 14 negative strand position 31,483,013 bp and ending after 31,482,624 bp (GRCm38/mm10). In addition there is a 9 bp insertion at the 390 bp deletion site (AGTGAGTGA) and 4 bp deletion (TAGT) 37 bp before the 390 bp deletion.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 37965 | TAA CAC ACA AGT CCC AAG TCC | Wild type Forward | A | |||
| 37966 | TCT ACT CTG CGG GCC TCA CT | Common | A | |||
| 37967 | ATG TAC TCC ACA CCT GAC ATG G | Mutant Forward | A | |||
| 37968 | Fluorophore-1 | TAG AGC AAG TTC TGA TAC ACC ACA TAC | Quencher-1 | WT Probe | ||
| 37969 | Fluorophore-2 | AAG AGC AAC CAT TCA AAG GGT | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 37965 | 0.40 uM |
| 37966 | 0.40 uM |
| 37967 | 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.