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Notes
This assay does not work well without the use of a Hotstart Taq polymerase.
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
3%
Mutant = 212 bp
Heterozygote = 212 bp and 297 bp
Wild type = 297 bp
Note: Stock# 012569 and Stock# 024109 have a mutant band size = 253 bp due to different polylinker length between primer annealing sites.
Separated by gel electrophoresis on a 1.5% agarose gel.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| oIMR9020 | AAG GGA GCT GCA GTG GAG TA | Wild type Forward | A | |||
| oIMR9021 | CCG AAA ATC TGT GGG AAG TC | Wild type Reverse | A | |||
| oIMR9102 | ACA TGG TCC TGC TGG AGT TC | Mutant Forward | A | YFP/EGFP | ||
| oIMR9103 | GGC ATT AAA GCA GCG TAT CC | Mutant Reverse | A | WPRE |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| oIMR9020 | 0.50 uM |
| oIMR9021 | 0.50 uM |
| oIMR9102 | 0.50 uM |
| oIMR9103 | 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 |
|---|---|
| 007903 | B6.Cg-Gt(ROSA)26Sortm3(CAG-EYFP)Hze/J |
| 024109 | B6.Cg-Gt(ROSA)26Sortm32(CAG-COP4*H134R/EYFP)Hze/J |
| 033760 | STOCK Braftm1Mmcm Gt(ROSA)26Sortm3(CAG-EYFP)Hze Nkx3-1tm4(cre/ERT2)Mms Ptentm1Hwu/AbshnJ |
| 033754 | STOCK Brca2tm1Brn Gt(ROSA)26Sortm3(CAG-EYFP)Hze Trp53tm1Brn Nkx3-1tm4(cre/ERT2)Mms Ptentm1Hwu/AbshnJ |
| 033753 | STOCK Gt(ROSA)26Sortm3(CAG-EYFP)Hze Brca1tm2Cxd Nkx3-1tm4(cre/ERT2)Mms Ptentm1Hwu/AbshnJ |
| 033761 | STOCK Gt(ROSA)26Sortm3(CAG-EYFP)Hze Krastm4Tyj Nkx3-1tm4(cre/ERT2)Mms Ptentm1Hwu/AbshnJ |
| 033757 | STOCK Gt(ROSA)26Sortm3(CAG-EYFP)Hze Nkx3-1tm4(cre/ERT2)Mms Ptentm1Hwu Tg(ARR2/Pbsn-MYC)7Key/AbshnJ |
| 033751 | STOCK Gt(ROSA)26Sortm3(CAG-EYFP)Hze Nkx3-1tm4(cre/ERT2)Mms Ptentm1Hwu/AbshnJ |
| 033759 | STOCK Gt(ROSA)26Sortm3(CAG-EYFP)Hze Nkx3-1tm4(cre/ERT2)Mms Smad4tm2.1Cxd Ptentm1Hwu/AbshnJ |
| 033750 | STOCK Gt(ROSA)26Sortm3(CAG-EYFP)Hze Nkx3-1tm4(cre/ERT2)Mms/AbshnJ |
| 033755 | STOCK Gt(ROSA)26Sortm3(CAG-EYFP)Hze Trp53tm1Brn Nkx3-1tm4(cre/ERT2)Mms Ptentm1Hwu/AbshnJ |
| 033756 | STOCK Gt(ROSA)26Sortm3(CAG-EYFP)Hze Trp53tm1Brn/Trp53tm3Tyj Nkx3-1tm4(cre/ERT2)Mms Ptentm1Hwu/AbshnJ |
| 12 strains use this protocol | |