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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
>chr8:126940894-126940992 99bp TGATTGGATTTTCCTTTCTTCC TGGTTCTTTTGCTACTGTGCTG
Mut= 90 bp
Wt= 99 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
CTGGAATCTGTATATTTCAGTAATAATGGCTTAATTATTTTATTTAGAAAGGGGGCTTTGTTCTGCTGCTGCACAGCTGTTGGGACAGTCTCTGTCTTCTGAATTGTTGGACCTGGGGATGCAGAAGAAAAGAGAAAGATCCTACCTAGAAACACCAGTTCACTCGCCAGGGAGgcagggattctggaggcaggaacgttttgtagaattaatgatggttgcagcaattagatgtcttgatccttaatgatgatagccaagtttttaatttatttttcttgtaggaagaaagaaacagaagcttgctaaggagagagctgggctttccaaggtaatgaaaatagtcttgtgtttttgcgtgtagattgcatgctctctaagcctcctgtgttttcctttggtggtggtaagagcaaatatgttaattgggtaagcagcaagatgagagtctccagagatggacatgaagagttaaagttctcggctacttaaaaatggttttaaaaaatacagcatttgattggattttcctttcttccttccgacagtaacctttaatcctagtttgagAAGCTCTGTTAGAGGCAGCCTGGGCAGCACAGTAGCAAAAGAACCACGTGTGTTAGTCCCACAGATCTTTAAGGTTTTTACACCTATGGATCAGAATTATATCAGGTTCTGACATTTAAAAAAACAAATTGTAATTTGTAGAATATAAATTTTCATTTTTTTCTTTTCATAGGTCCTTAGTTTACAATTTTTAATGACTGTAGGTTTTTATCTACTTAATT
This mutation is a 396 bp deletion beginning at Chromosome 8 position 126,940,940 bp and ending after 126,941,335 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 38903 | TGA TTG GAT TTT CCT TTC TTC C | Wild type Forward | A | |||
| 38904 | TGG TTC TTT TGC TAC TGT GCT G | Common | A | |||
| 38905 | AGA GAA AGA TCC TAC CTA GAA ACA CCA | Mutant Forward | A | |||
| 38906 | Fluorophore-1 | TCC GAC AGT AAC CTT TAA TCC TAG TT | Quencher-1 | WT Probe | ||
| 38907 | Fluorophore-2 | ACT CGC CAG GGA GAA GCT | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 38903 | 0.40 uM |
| 38904 | 0.40 uM |
| 38905 | 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.