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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 = ~300 bp
Heterozygote = ~300 bp and 603 bp
Wild type = 603 bp
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 |
|---|---|---|---|---|---|---|
| oIMR8052 | GCG AAG AGT TTG TCC TCA ACC | Mutant Reverse | A | SA site | ||
| oIMR8545 | AAA GTC GCT CTG AGT TGT TAT | Common | A | ROSA26 | ||
| oIMR8546 | GGA GCG GGA GAA ATG GAT ATG | Wild type Reverse | A | ROSA26 |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| oIMR8052 | 0.50 uM |
| 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 |
|---|---|
| 003310 | 129S-Gt(ROSA)26Sortm1Sor/J |
| 007844 | 129S4/SvJae-Gt(ROSA)26Sortm2(FLP*)Sor/J |
| 007689 | 129S4/SvJaeSor-Gt(ROSA)26Sortm4(attB/attP)Sor/J |
| 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 |
| 011008 | B6.129P2(Cg)-Gt(ROSA)26Sortm1(tTA)Roos/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 |
| 012930 | B6.129S4-Gt(ROSA)26Sortm2(FLP*)Sor/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 |
| 006965 | B6.Cg-Gt(ROSA)26Sortm1(rtTA*M2)Jae/J |
| 008883 | B6;129-Gt(ROSA)26Sortm1(SNCA*A53T)Djmo/TmdJ |
| 008889 | B6;129-Gt(ROSA)26Sortm2(SNCA*119)Djmo/TmdJ |
| 029415 | B6;129S4-Gt(ROSA)26Sortm1(rtTA*M2)Jae Col1a1tm1(tetO-cas9)Sho/J |
| 016836 | B6;129S4-Gt(ROSA)26Sortm1(rtTA*M2)Jae Col1a1tm7(tetO-HIST1H2BJ/GFP)Jae/J |
| 003309 | B6;129S4-Gt(ROSA)26Sortm1Sor/J |
| 007900 | C57BL/6-Gt(ROSA)26Sortm1(HBEGF)Awai/J |
| 009427 | FVB.129S4(B6)-Gt(ROSA)26Sortm1Sor/J |
| 006331 | STOCK Gt(ROSA)26Sortm1(DTA)Jpmb/J |
| 011004 | STOCK Gt(ROSA)26Sortm1(rtTA*M2)Jae Col1a1tm3(tetO-Pou5f1,-Sox2,-Klf4,-Myc)Jae/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 |
| 30 strains use this protocol | |