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Protocol 37500: Probe Assay - H2-K1<b-tm1Bpe> Probe (NOD/NSG Only) Probe Alternate5
Version 1.0
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= 96 bp
Wild Type = 78 bp
Sequence
Wt (NOD background) Sequence: GACCGCGGGGTCCCGCAGGCTCTCACACGTTCCAGCGGATGTTCGGCTGTGACGTGGGGTCGGACTGGCGCCTCCTCCGCGGGTACCAGCAGTTCGCCTACGACGGCCGCGATTACATCGCCCTGAACGAAGACCTGAAAACGTGGACGGCGGCGGACACGGcggcgctgatcaccagacGCAAGTGGGAGcaggctggtgatgcagagtattacAGGGCCTACCTAGAGGGCGAGTGCGTGGAGTGGCTCCGCAGATACCTGGAGCTCGGGAATGAGACGCTGCTGCGCACAGGTGCAGGGGCCGCGGGCAGCTCCTCCCTCTGCCCTCGGGCTGGGGCTCAGTCCTGGGGAAGAAGAAACCCTCAGCTGGGTGATGCCCCTGTCTCAGAGGGGAGAGAGTGTCCCTGGGTCTCCTGATTCCTCATCACAGGGACTGCACTGAGTCTCCCAGGACTCAGCCCTCTCCCTGGACAGTGCCCAGGCTGTCTCAGGAGGGAAGGAGAGAATTTCCCTGAGGTAACAACAGCTGCTCCCTTCAGTTCCCCTGCAGCCTCTGTCAGCCATGGCCTCTCCCAGGCCGG
Mutant Sequence: ctttgctttccCtggttaagcagtacagccccaaaatggttaaggttgcaagCTTGCTGGTGAAAAGGACCTCTCGAAGTGTTGGATACAGGCCAGACTttgttggatttgaaattccagacaa
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 49452 | AGC AGT ACA GCC CCA AAA TG | Mutant Forward | A | |||
| 49453 | CTG GCC TGT ATC CAA CAC TTC | Mutant Reverse | A | |||
| 49454 | Fluorophore-1 | AGG TTG CAA GCT TGC TGG TG | Quencher-1 | MUT Probe | ||
| 50991 | CTG AAC GAA GAC CTG AAA ACG | Wild type Forward | A | |||
| 51486 | CCT GTA ATA CTC TGC ATC ACC AG | Wild type Forward | A | |||
| 51487 | Fluorophore-2 | CGG CGC TGA TCA CCA GAC | Quencher-2 | WT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 49452 | 0.40 uM |
| 49453 | 0.40 uM |
| 50991 | 0.40 uM |
| 51486 | 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.