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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
>chr19:60209892-60209974 83bp AAGTTCCTACTGACATTCTTGAGC CCCTGATTTTCTTAAGTATGGACTC
Mutant= 91 bp
Wild Type = 83 bp
Sequence
Wt Sequence (deletions in lower case)
GGCCCTGTCTTAGTAAAAGAACAAAACAAAATAAAAATTTAAAACTACATTGTTTTAAGTTCCTACTGACATTCTTGAGCCATGAGTGCTTTACTGAgcgtcgtttaggcagaagagtccatacttaagaaaatcagggttctgttttatccattttagattttaaagtttaaattggctgtccttaggtcattggcagacatattggtctcctgtggtatctgttagtggttttaaaatagtttccatggctactctttgagtttgaattcatccttccctttctcctattagaagaaaagatgtggagtgggctgttacctcctggactaaatgaaagtgatgttgaatcagattctgaggatgaaatcaaactagagaacccagaaccttcagaacataatttacaggaagatggaaagactggcagcagtacgaagccagcagtgtcagacttcccgacaggccagccagagacggagacagaggctgatgcggatgcatatgagaagtgtccttctgggattcccttaaacatatggaatgtaggtttccctttgtgtggttaggaaagtatgcatgttcatgtgttctctgtgctgtgggcaaagtatgttgggatataacagacagtagaggggcctcagaacagattctactgttggatactagttttattttacataagtagaagtagtaaaaccttcataggattatctacttcataggattatctacctaacatgataaaggagtttatttctttatttcttcctttaattatatgagttattgagcttcattctccactatcaaacagcttatatcaaaaccagtgaatgccccaattcagctgagagttgtgaggactaaagtatttcaagaacagaattccttagttggtggctttagaaatggcagagcataaggatttctatttaacatgtctgttcctttttaaactgtatgtttatcttcagtcacacacacacacacacacatcttttctccttttacatagaaatttcaagaattgcataaaaaaaattctgaacagaaaaattcaacgccaagattcagacagaaaaaaagaaaacgctccaaaaaaggtgtttatttttaaatttagattgttagttatgctaactcttaagttgattaattgaattaatttcttaaaacatgattttgaaataatagctgcataattaagtattgacactctaaatttcttttaggcaaattgaagaatgaaaaggaatctcacaggtaggtagattttaaagtagttactattcagcagtgttgtggGATTTTCTAGATTGCTGCACTACTTACCTTTG
This mutation is a 1214 bp deletion beginning at Chromosome 19 position 60,220,288 bp and ending after 60,221,501 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 65996 | AAG TTC CTA CTG ACA TTC TTG AGC | Common | A | |||
| 65997 | CTC ATG TCT GAT CAT TAC CAA AGG | Mutant Reverse | A | |||
| 65998 | CCC TGA TTT TCT TAA GTA TGG ACT C | Wild type Reverse | A | |||
| 65999 | Fluorophore-1 | CTG AGC GTC GTT TAG GCA G | Quencher-1 | WT Probe | ||
| 66000 | Fluorophore-2 | AGT GCT TTA CTG AGA TTT TCT AGA TTG CT | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| 65996 | 0.50 uM |
| 65997 | 0.50 uM |
| 65998 | 0.50 uM |
| Glycerol | 6.50 % |
| Dye | 1.00 X |
| Kapa 2G HS taq polym | 0.03 U/ul |
| 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.