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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
Mutant= 111 bp
Wild Type = 110 bp
>chr8:105355380+105355489 110bp CTGGGGAGGAAATGTCTGAG TGCAGACCTTGTACAGCACCT
Sequence
Sequence (deleted region in lower case, ^x^ is an insertion)
CCCTGTGGCCGTGTTGGCCGTCTTTGAGGAGGTGCATGTCAATCAGACCCTCTTTATCATCATCTTCGGCGAGTCTCTGCTCAATGATGCAGTAACTGTGGTAAGAACATCTGGTGGCTTGCTGACCCCTGACCTTAGGCGTGACCGGGCTTAGACCTTTCCTCCTAGTCCCCTGGGGAGGAAATGTCTGAGCTTTCTTCTGTTCCCGCCCACTTCCCCC^ag^gtatggccccctcctacttcctgtctcccctcttgtcactcaggtgctgtacaaggtctgcaactcctttgtggagatgggctctgccaacgtgcaggccactgactacctaaagggcgtcggtcagtatttcccccaacttggcctgcattcaatgtctgccttctctggattgttgcgactcacccagccagcgtttgggacaGAGGAGGCTATTCCGATGTCTCATTCCTTCCTCTCCTCTATGGAGAAATGGGGTCTGGGAGGAGCTTGCCTTGGAATTCTAGTCTGGGATCCTGAGCCCCAGGGAGTGTGGTGGGTGCCTTCCTCTCTATTTCTTACTGGGCAACAGACTTTGTCAGCCCAGCTTGGGGAGGGTCTGGGCCAGGGGTCGTGCTGACAACCCACTCCTCCCCCAGCCTCCCTGTTTGTGGTCAGTCTGGGCGGGGCAGCGGTGGGCTTAGTCTTTGCCTTCCTCCTGGCCCTGACCACACGCTTCACCAAGCGGGTCCGCATCATCGAGCCGTTGCTGGTCTTCCTCCTCGCCTACGCAGCCTACCTCACTGCTG
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 37753 | CTG GGG AGG AAA TGT CTG AG | Common | A | |||
| 37754 | CTC CCA GAC CCC ATT TCT C | Mutant Reverse | A | |||
| 37755 | TGC AGA CCT TGT ACA GCA CCT | Wild type Reverse | A | |||
| 37756 | Fluorophore-1 | AGG CTA TTC CGA TGT CTC ATT C | Quencher-1 | MUT Probe | ||
| 37757 | Fluorophore-2 | CCT ACT TCC TGT CTC CCC TCT T | Quencher-2 | WT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 37753 | 0.40 uM |
| 37754 | 0.40 uM |
| 37755 | 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.