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Notes
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
Sequence
Mut = T H2<k> Jrs: 646, 656, 465, 4804
WT= C other haplotypes
tcccaccaTGAGCCTCTACCCTCTAGGAATTCG(c/t)CAGGGACCAGTCAGGTC
ACTCAGCTggggcttct
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 21918 | Fluorophore-1 | TAG GAA TTC GTC AGG GAC CAG | Quencher-1 | MUT Probe | ||
| 21919 | Fluorophore-2 | AGG AAT TCG CCA GGG ACC | Quencher-2 | WT Probe | ||
| 22189 | ATC CCA CCA TGA GCC TCT AC | Forward | A | |||
| 22190 | CAG CTG AGT GAC CTG ACT GG | Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 22189 | 0.40 uM |
| 22190 | 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
| Stock Number | Strain Name |
|---|---|
| 000465 | B10.BR-H2k2 H2-T18a/SgSnJ |
| 004804 | B10.BR-H2k2 H2-T18a/SgSnJJrep |
| 036602 | B10.Cg-H2k2 H2-T18a Tg(SV40-HA)HA104Ajca/VradMmjax |
| 000666 | C57BL/10SnJ |
| 000486 | MRL/MpJ |
| 026243 | NOD.Cg-(D9rs4135590-D9rs13480186)H2k2Tg(ILK3mHEL)3Ccg Tg(TcrHEL3A9)1Mmd/SlsgJ |
| 6 strains use this protocol | |