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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
>chr10:56196823-56196927 105bp AAGTGTTTGGTCTGTGCTGCT GGTGAGAGGGTAAAAGTCTGTG
Mut= 107 bp
Wt= 105 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletions in lower case; and insertion with carrots ^g^ (T insertion):
TCAAAATGTAGTGTTGCCTACTGTGCCACTGTTGGATGGCACCATTAAAGCCTCCAGCACCTCTGTGCAAGCCTGCAGCTTCATTTCACAGCACCACGTGACCCTTCCTCCCGCCACCATTCACTCCTACTTCCTGCTGTGACCAGATTTCACAGCAGTGCATTTTGTTGTTGGTTTTACCCTTAGTTGTGGATAATTTTACTTTTGGCTAAATGAAGACATGCCCTTTAGAATAGACACGGCCTTCCCCTGG^tttctgcgt^AGTGTATCTTGTGTTTATTTTCTGTTCCATGCGAGAAGTGTTTGGTCTGTGCTGCTGTAAGATCGCAAATGAGGATGGGCTGTTttgcttccctaggaccggattttaaagttctacgcacagacttttaccctctcaccgttacatatgaccaaggagatttatacaagcttaggtatgttcaccaaatgacttcaattgttgttctcctatatgcacttaaaatcccgtaagaaatgacttcttccagaagcattgcgtaatatatataacgactgtatattttccctttccttttcagctaaatatctggaggtatattttctttctagagaaaatcaccttcctactctttcaactggagtagacataaccagtcccaatgtgacccgcttactgaaaaaggtatccacttactatgatgtactgtatggaaagtagggctatatttaagaattaaactcttttgagagcttttgtatactgcttataggttcgccttctgaatgagtaccagaaagaggctccatctttttggattcgtcacccagagaagtaagttccccaactcctgtgatgtaaggtataggctatgtccttcaattatttcaaggacagaaggaacagtagtcttgagcgatGGACATGGTTCTATATGCCACTTAGTTTTTGTCAGCTTGACACCAAATAGGACCACCTAGGAAATAGACCCTCATCTGAGGAACTGCCCTCATCACTTGCCTATGTGTGTGTTTTTGTAACTGTAAATTGATATAGGGGGCCTCCCAGGGGCAATACCTTCTGTAGGCACATGGCCCTGGGCTGTATAAGAAAAGTAGCAAGCTTGAGGAGGCAAGACAGCAAGCAACGCCTCAATGGTTTTTGCCCCAAATTCCTGTCTTGAGCTCCTGGTCTGGCTTCCCTT
This mutation is a 557 bp deletion beginning at Chromosome 10 position 56,196,322 bp and ending after 56,196,878 bp (GRCm38/mm10). In addition, there is a 1 bp (T) insertion and a 9 bp deletion (ACGCAGAAA) 84 bp after the deletion.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 48684 | AAG TGT TTG GTC TGT GCT GCT | Common | A | |||
| 48685 | GGT GAG AGG GTA AAA GTC TGT G | Wild type Reverse | A | |||
| 48686 | AGG TGG TCC TAT TTG GTG TCA | Mutant Reverse | A | |||
| 48687 | Fluorophore-1 | CTT CCC TAG GAC CGG ATT TTA A | Quencher-1 | WT Probe | ||
| 48688 | Fluorophore-2 | ATG GGC TGT TGG ACA TGG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 48684 | 0.40 uM |
| 48685 | 0.40 uM |
| 48686 | 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.