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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= 93 bp
Wild Type = 94 bp
This assay may be capable of distinguishing hemi from hom. Transgene insertion site is known to be on mouse Chr 14.
This assay is designed around this insertion site, but it has not been tested on hom animals.
This assay is NOT able to be used for copy number evaluation. If this is required, it is suggested to type by qPCR.
This protocol can be used for conventional PCR. Omit probes and run as a separated assay.
Sequence
Wt Sequence:
gttcatatcttgaagttctcactcctattatgatgatatttggaggctgagcaTttgagaggtgaggtttatggtgaCataggagaaggattgaatgggatcacatagtgcccttaaaagaagcagtacagagagctaccctcctctgaacagtccctatacactaga
Mutant Sequence:
TATTCTGAATCTTAGAGTTTTTATTTGTTGAAATTTTGGACTGGATCTGTGACTTTCCAAAGTTCATATCTTGAAGTTCTCACTCCTATTATGATGATATTTGGAGGCTGAGCATTGAGAGGTGAGGTTTATGGTGatGCTGAAGGTCGCGCGCATTCCCGATGAAGAGGCCGGTTACCGCCTGTTGACATGGTG
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 40119 | TAT TAT GAT GAT ATT TGG AGG CTG A | Common | A | mChr14 | ||
| 40120 | TTT TAA GGG CAC TAT GTG ATC C | Wild type Reverse | A | mChr14 | ||
| 40121 | Fluorophore-1 | TTT ATG GTG ACA TAG GAG AAG GAT TG | Quencher-1 | WT Probe | ||
| 40122 | GGT AAC CGG CCT CTT CAT C | Mutant Reverse | A | vector | ||
| 40123 | Fluorophore-2 | TAT GGT GAT GCT GAA GGT CG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 40119 | 0.40 uM |
| 40120 | 0.40 uM |
| 40122 | 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.