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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
AAGCAGACACAGGAGAGAGGATGTTCTGTaaaacaagcatgtga
Aaggatgcg(A/-)tg(CGTG/-----)atgaaggattctttgctaaaGACACA
ATTATGGATCTGCAA
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 21943 | Fluorophore-1 | ATG CGA TGC GTG ATG AAG | Quencher-1 | MUT Probe | ||
| 21944 | Fluorophore-2 | AAG GAT GCG TGA TGA AGG AT | Quencher-2 | WT Probe | ||
| 22205 | CAC AGG AGA GAG GAT GTT CTG | Forward | A | |||
| 22206 | AGG GTT GCA GAT CCA TAA TTG | Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 22205 | 0.40 uM |
| 22206 | 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 | -- | repeat steps 2-3 for 40 cycles |
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 |
|---|---|
| 002845 | C.B-H2b Tg(H2-Dd)D8Gja/LilJ |
| 001952 | C.B10-H2b/LilMcdJ |
| 000438 | C3.SW-H2b/SnJ |
| 000666 | C57BL/10SnJ |
| 004353 | C57BL/6-Tg(UBC-GFP)30Scha/J |
| 000664 | C57BL/6J |
| 005304 | C57BL/6NJ |
| 007076 | CByJ.B6-Tg(UBC-GFP)30Scha H2b/J |
| 002591 | NOD.B10Sn-H2b/J |
| 039016 | NOD.Cg-H2b Commd10Tg(Vav1-icre)A2Kio/JkraJ |
| 039015 | NOD.Cg-Myd88tm1.1Defr H2b/JkraJ |
| 039017 | NOD.Cg-Myd88tm1Defr H2b/JkraJ |
| 039018 | NOD.Cg-Myd88tm1Hlz H2b/JkraJ |
| 13 strains use this protocol | |