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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
>chr5:3595585-3595734 150bp ACAATAGCGCTTCTCACAGG TCTGCCTAAAGACTACCACACTCC
Mut = 116 bp
Wt = 150 bp
Fam = Mut
Hex = Wt
Sequence
Wt Sequence (deletions in lower case):
ATACCGGGAGGGCCGACGCCCTCGCGCGGTCAAGGTAGAGTGACTCGGATTCTTCTCTCCCCTCGGCAGGGACACTAAATTCCTGTGAGCACTAGCTCCCCAAGGCCTGCCTCCATAAAGGCATGGCAGGGATGTTGTGAGATCCATCTCTAAGTGGAGGGGGCACGAAGAACCCGGGCCCCGGTACCAAAGGTTATCACTACCCTCGGTTAGCTGCTTACCAAACCTCCAACCCACTATTTTTAGCTTTGTATCCTATGTGTTTACAATAGCGCTTCTCACAGGGCaagcccagggtgagtgagtgttcagtggctgtttacattaacctctctggagtcagcttttggaaaagacgtctgttaagtggtttagtcagatacccttatgtggagtgtggtagtctttaggcagaagagtgacagtttagtgtgtatgttttgcagaccggattgtttcttgttttcaaagctccatttaaaagggggtgggaggaccgatttgccataggcttgtttgaagagtacatttaacgaggtctcctttgactttaggtatatacaatcaatttggagtctcagtacttactaatacaaggagttcctgcagtgggagccatgaaggaattagttgagcgttttgctctgtatggtgcgattgaacagtataacgctctagatgaatacccagcggaagattttacagaagtttatctcattaaatttgtgaaattacagagtgcaaggtatgtacaagttaagcagaactcctgttaaaatcttattcctgttgctgtcattttggccccaactgtttcccaaatgTACCCAAAGCACAGAAGCACTTCTTGCTCTCAGTCTTCCTGATTGGGGGGAGGAGGGGAGGGGACAGGGTTTATTTGGATAGCCTTAGCTGTGCTAGAATTAGTTCTGTAGACCAGGCTGGCCTTGAACTCAAAGATCCCTTTGCCTTTGCCTCTGACTCTCGAGTGCTGGGTTTAAAGGTATGCACCACTCCACCTCGCCTTCCAGTTTGTTTTTCACCAGGCCTCACATCTGCGACTTAATCACTCTAACAGTGA
This mutation is a 427 bp deletion beginning at Chromosome 5 position 3,595,181 bp and ending after 3,595,707 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 49315 | ACA ATA GCG CTT CTC ACA GG | Common | A | |||
| 49316 | TCT GCC TAA AGA CTA CCA CAC TCC | Wild type Reverse | A | |||
| 51231 | Fluorophore-1 | CTT CCT GAT TGG GGG GAG G | Quencher-1 | MUT Probe | ||
| 51232 | Fluorophore-2 | AGG GTG AGT GAG TGT TCA GTG G | Quencher-2 | WT Probe | ||
| 51931 | GGC TAT CCA AAT AAA CCC TGT | Mutant Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| 49315 | 0.50 uM |
| 49316 | 0.50 uM |
| 51931 | 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.