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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
Mutant (embryonic lethal) = 100 bp
Heterozygote = 100 bp and 250 bp
Wild type = 250 bp
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 22907 | TGT CTT TCC CCA GCA CAG T | Wild type Forward | A | |||
| 22908 | CTG CAT AGT ACG CTA TAC CCT GT | Common | A | |||
| oIMR9592 | GCA GGT CGA GGG ACC TAA TA | Mutant Forward | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| 22907 | 0.50 uM |
| 22908 | 0.50 uM |
| oIMR9592 | 0.50 uM |
| Glycerol | 6.50 % |
| Dye | 1.00 X |
| Kapa 2G HS taq polym | 0.03 U/ul |
| DNA |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 94.0 | -- | |
| 2 | 94.0 | -- | |
| 3 | 65.0 | -- | -0.5 C per cycle decrease |
| 4 | 68.0 | -- | |
| 5 | -- | repeat steps 2-4 for 10 cycles (Touchdown) | |
| 6 | 94.0 | -- | |
| 7 | 60.0 | -- | |
| 8 | 72.0 | -- | |
| 9 | -- | repeat steps 6-8 for 28 cycles | |
| 10 | 72.0 | -- | |
| 11 | 10.0 | -- | hold |
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
JAX uses a 'touchdown' cycling protocol and therefore has not calculated the optimal annealing temperature for each set of primers.
Strains Using This Protocol
| Stock Number | Strain Name |
|---|---|
| 008180 | 129S/Sv-Krastm4Tyj/J |
| 033964 | B6.129(Cg)-Ptf1atm2(cre/ESR1)Cvw Krastm4Tyj Ptentm1Hwu/J |
| 032435 | B6.129-Krastm4Tyj Trp53tm1Brn/J |
| 008179 | B6.129S4-Krastm4Tyj/J |
| 035169 | B6.Cg-Krastm4Tyj Apctm1Tno Tg(CDX2-cre/ERT2)752Erf/MaraJ |
| 019104 | B6N.Cg-Krastm4Tyj/CjDswJ |
| 032429 | STOCK Krastm4Tyj Trp53tm1Brn Tg(Pdx1-cre/Esr1*)#Dam/J |
| 7 strains use this protocol | |