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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
Mutant = ~500 bp
Heterozygote = ~500 bp and 367 bp
Wild type = 367 bp
Heterozygote = ~500 bp and 367 bp
Wild type = 367 bp
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 9414 | TTC AGA CAC TGC GTG ACT | Mutant Forward | A | birA | ||
| 9415 | GGC TCC AAT GAC TAT TTG C | Mutant Reverse | A | birA | ||
| 9416 | GTG TAA CTG TGG ACA GAG GAG | Wild type Forward | B | |||
| 9417 | GAA CTT GAT GTG TAG ACC AGG | Wild type Reverse | B |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTPS-kapa | 0.26 mM |
| 9414 | 0.50 uM |
| 9415 | 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.
Reaction B
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTPS-kapa | 0.26 mM |
| 9416 | 0.50 uM |
| 9417 | 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 |
|---|---|
| 010920 | FVB;129P2-Gt(ROSA)26Sortm1.1(birA)Mejr/J |
| 018121 | STOCK Gt(ROSA)26Sortm1(birA)Mejr Gata4tm3.1Wtp/J |
| 025980 | STOCK Gt(ROSA)26Sortm1.1(birA)Mejr Ep300tm1.1Wtp/Mmjax |
| 025983 | STOCK Gt(ROSA)26Sortm1.1(birA)Mejr Mef2ctm1.1Wtp/Mmjax |
| 025978 | STOCK Gt(ROSA)26Sortm1.1(birA)Mejr Nkx2-5tm1.1Wtp/Mmjax |
| 025979 | STOCK Gt(ROSA)26Sortm1.1(birA)Mejr Srftm1.1Wtp/Mmjax |
| 025981 | STOCK Gt(ROSA)26Sortm1.1(birA)Mejr Tead1tm1.1Wtp/Mmjax |
| 025977 | STOCK Gt(ROSA)26Sortm1.1(birA)Mejr Zfpm2tm1.1Wtp /Mmjax |
| 025982 | STOCK Rbpjtm1.1Wtp Gt(ROSA)26Sortm1.1(birA)Mejr/Mmjax |
| 9 strains use this protocol | |