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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
Melt Peaks
Wt = 88°C +/- 1
Mut = 88°C +/- 1
Wt = 88°C +/- 1
Mut = 88°C +/- 1
Mutant = 340 bp
Heterozygote = 340 bp and 650 bp
Wild type = 650 bp
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| oIMR8052 | GCG AAG AGT TTG TCC TCA ACC | Mutant | A | SA site | ||
| oIMR8545 | AAA GTC GCT CTG AGT TGT TAT | Common | A, B | ROSA26 | ||
| oIMR8546 | GGA GCG GGA GAA ATG GAT ATG | Wild type | B | ROSA26 |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTPS-kapa | 0.26 mM |
| oIMR8052 | 0.50 uM |
| oIMR8545 | 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 |
| oIMR8545 | 0.50 uM |
| oIMR8546 | 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 |
|---|---|
| 009582 | B6(129S4)-Et(cre/ERT2)1645Rdav/J |
| 009585 | B6(129S4)-Et(cre/ERT2)2047Rdav/J |
| 012687 | B6(129S4)-Tg(SYN1-icre/mRFP1)9934Rdav/J |
| 012580 | B6.129P2(Cg)-Hprt1tm59(Ple123-lacZ)Ems/Mmjax |
| 012581 | B6.129P2(Cg)-Hprt1tm62(Ple153-lacZ)Ems/Mmjax |
| 012583 | B6.129P2(Cg)-Hprt1tm68(Ple127-lacZ)Ems/Mmjax |
| 013586 | B6.129P2-Gt(ROSA)26Sortm1Nik/J |
| 003474 | B6.129S4-Gt(ROSA)26Sortm1Sor/J |
| 007743 | B6.129S4-Gt(ROSA)26Sortm3(phiC31*)Sor/J |
| 032087 | B6.129S6(Cg)-Gt(ROSA)26Sortm1(DTA)Jpmb/J |
| 014602 | B6.Cg-Gt(ROSA)26Sortm1(rtTA*M2)Jae Col1a1tm1(tetO-mCherry)Eggn/J |
| 023749 | B6.Cg-Gt(ROSA)26Sortm1(rtTA*M2)Jae Tg(tetO-Pou5f1,-Sox2,-Klf4,-Myc)1Srn/J |
| 010527 | B6;129-Gt(ROSA)26Sortm1(DTA)Mrc/J |
| 008883 | B6;129-Gt(ROSA)26Sortm1(SNCA*A53T)Djmo/TmdJ |
| 008889 | B6;129-Gt(ROSA)26Sortm2(SNCA*119)Djmo/TmdJ |
| 022516 | B6;129S-Gt(ROSA)26Sortm1(Cdkn1c)Jfpa/J |
| 003309 | B6;129S4-Gt(ROSA)26Sortm1Sor/J |
| 019101 | B6N.129S4(B6)-Gt(ROSA)26Sortm1Sor/CjDswJ |
| 019013 | B6N.129S6(Cg)-Gt(ROSA)26Sortm2(EGFP/cre)Alj/J |
| 007900 | C57BL/6-Gt(ROSA)26Sortm1(HBEGF)Awai/J |
| 011013 | STOCK Gt(ROSA)26Sortm1(rtTA*M2)Jae Col1a1tm5(tetO-Pou5f1,-Klf4,-Myc)Jae/J |
| 018903 | STOCK Gt(ROSA)26Sortm2(EGFP/cre)Alj/J |
| 012584 | STOCK Hprt1tm69(Ple134-lacZ)Ems/Mmjax |
| 23 strains use this protocol | |