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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
>chr2:181696592+181696684 93bp AGGCTCATCCCTGTGCTTT GGCACTCCTTTCTCCCAAC |
Mutant= 99 bp
Wild Type = 93 bp
Sequence
Wt Sequence (deletions in lower case):
AGGCTCATCCCTGTGCTTTTCCACACTAGTAACCTCCCACATCTAATGGCACTgaagggcaccacttgcaggctgttgggagaaaggagtgcccacccaccaacatctccttgcttctgtggctgtggctgaaggagaggcagcatccatgtttgtcttttcctgtgggggcccagggggaaaagaggggaaataccagagaacccttggaagagaactttatatgagagggtggggagctgctgtaaccgtagggtctctgaacgccaaggtctcagtcatcgggggaagatgttgccatctacctcctcttccaggattgtctcaacacgcctccgctgcagagagagaggctggagccaatgtgtggcgaggcacggaaccctcccacccgtctccggggttccccacacctcgtgtcggtccagaggatcagggatctttgtgggcttcagttggggtggtaggaacagttccagtcggatggcagcgcgcgcggagctctgcttctggatcagcaaagagatttcctctgcctgggaaccaagtttactgagcaaccctgcgagcatgctagaggagtaccctccctccacccgcagggtaggccctctttagaagatgcactgggtcctctcccttcttcctacccgcattcgggtgtagcgcaaccgcagggtatggactcggccccgggcttccgcggccttaataaggcccagaaaccgattttgatgctcagatggtagatttaatgcctgtgaccaggtgcgcgggtcaggcatgtcgtagggtaactccaccgactcgcgtggaaacatggaggccaccttgcctatgtcgtcaagaaagtcgccaaagagcaggtggcgttggcgtagtgtaggggacaaggctaccagcctctgcttagtcaatgcccagcctgtcttcggagggtctgtagtcctcgggcccttcctgtgcttctctccagctccagctccagctccagctccagctccagctccagatgcactgtttttgtcccgctcccgtgcaccctttactcccagggtcggcatgctccggtctgtggaagaaaaggggatgtccagccacgtaggggaaaactatggctccagttctgtatctatgggacttcctgtagccacctaactcatattcctaaactggtagctgaccctgtgcatatctgcagtaggccatgactttctgatcctctccacttttaggagtatacccaatttaggactataaaacccgaCGACGGATTGTACTACCTGCTATTGTTGCTCCACAATCCACATCAC
This mutation is a 1214 bp deletion beginning at Chromosome 2 position 181,696,645 bp and ending after 181,697,858 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 62674 | AGG CTC ATC CCT GTG CTT T | Common | A | |||
| 62675 | GGC ACT CCT TTC TCC CAA C | Wild type Reverse | A | |||
| 62676 | GTG ATG TGG ATT GTG GAG CA | Mutant Reverse | A | |||
| 62677 | Fluorophore-1 | ATC TAA TGG CAC TGA AGG GCA C | Quencher-1 | WT Probe | ||
| 62678 | Fluorophore-2 | AAT GGC ACT CGA CGG ATT GTA C | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 62674 | 0.40 uM |
| 62675 | 0.40 uM |
| 62676 | 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.