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Notes
This assay will NOT distinguish hemizygous from homozygous
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
Transgene = 89 bp
Internal positive control = 74 bp
Sequence
Tg Sequence:
ATGCTCCTGTCTGTGTGCAGATCCTGGGCTGCCTGGTGCAAGCTGAACAACAGGAAATGGTTCCCTGCTGAACCTGAGGATGTGAGGGA
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 22926 | TCC CTC ACA TCC TCA GGT TC | Transgene Reverse | A | |||
| 41665 | ATG CTC CTG TCT GTG TGC AG | Transgene Forward | A | |||
| 41666 | Fluorophore-1 | TGC CTG GTG CAA GCT GA | Quencher-1 | Tg Probe | ||
| oIMR1544 | CAC GTG GGC TCC AGC ATT | Internal Positive Control Forward | A | |||
| oIMR3580 | TCA CCA GTC ATT TCT GCC TTT G | Internal Positive Control Reverse | A | |||
| TmoIMR0105 | Fluorophore-2 | CCA ATG GTC GGG CAC TGC TCA A | Quencher-2 | IC Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 22926 | 0.40 uM |
| 41665 | 0.40 uM |
| oIMR1544 | 0.40 uM |
| oIMR3580 | 0.40 uM |
| Wt Probe | 0.15 uM |
| Mutant Probe | 0.15 uM |
| DNA |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 95.0 | -- | |
| 2 | 95.0 | -- | |
| 3 | 60.0 | -- | |
| 4 | -- | repeat steps 2-3 for 40 cycles | |
| 5 | 40.0 | -- | Forever |
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
Strains Using This Protocol
| Stock Number | Strain Name |
|---|---|
| 035764 | B6(129)-Jchainem1(icre/ERT2)Deep/J |
| 039826 | B6(129S4)-Fcgr1em1.1(icre)Moli/J |
| 035435 | B6(129S4)-Xcr1tm1.1(cre)Kmm/J |
| 009587 | B6(129S4)-Et(icre)1402Rdav/J |
| 009588 | B6(129S4)-Et(icre)1470Rdav/J |
| 009589 | B6(129S4)-Et(icre)1555Rdav/J |
| 009586 | B6(129S4)-Et(icre)754Rdav/J |
| 010696 | B6(129S4)-Et(icre/ERT2)10596Rdav/J |
| 010697 | B6(129S4)-Et(icre/ERT2)10727Rdav/J |
| 012689 | B6(129S4)-Et(icre/ERT2)14163Rdav/J |
| 012690 | B6(129S4)-Et(icre/ERT2)14208Rdav/J |
| 012694 | B6(129S4)-Et(icre/ERT2)14915Rdav/J |
| 012687 | B6(129S4)-Tg(SYN1-icre/mRFP1)9934Rdav/J |
| 037836 | B6(FVB)-Il1bem1Mora/J |
| 039650 | B6(SJL)-Cd40lgtm1.1(icre)Tega/J |
| 038129 | B6.Cg-Ascl3tm2.1(icre/ESR1*)Ovi/J |
| 008610 | B6.Cg-Commd10Tg(Vav1-icre)A2Kio/J |
| 038342 | B6.Cg-Gas1em1(icre/ERT2)Nono/J |
| 023677 | B6.Cg-Hprt1tm332(Ple272-icre/ERT2)Ems/Mmjax |
| 023690 | B6.Cg-Hprt1tm345a(icre/ERT2)Ems/Mmjax |
| 023691 | B6.Cg-Hprt1tm346a(Ple264-icre/ERT2)Ems/Mmjax |
| 023692 | B6.Cg-Hprt1tm347a(Ple155-icre/ERT2)Ems/Mmjax |
| 023693 | B6.Cg-Hprt1tm348a(Ple265-icre/ERT2)Ems/Mmjax |
| 023694 | B6.Cg-Hprt1tm349a(hs671-icre/ERT2)Ems/Mmjax |
| 023695 | B6.Cg-Hprt1tm350a(Ple232-icre/ERT2)Ems/Mmjax |
| 023696 | B6.Cg-Hprt1tm351a(Ple198-icre/ERT2)Ems/Mmjax |
| 023697 | B6.Cg-Hprt1tm352a(Hspa1a-icre/ERT2)Ems/Mmjax |
| 023698 | B6.Cg-Hprt1tm353(Ple274-icre/ERT2)Ems/Mmjax |
| 023699 | B6.Cg-Hprt1tm354(Ple283-icre/ERT2)Ems/Mmjax |
| 023705 | B6.Cg-Hprt1tm360a(Ple253-icre/ERT2)Ems/Mmjax |
| 023706 | B6.Cg-Hprt1tm361a(Ple267-icre/ERT2)Ems/Mmjax |
| 023708 | B6.Cg-Hprt1tm363a(hs1218-icre/ERT2)Ems/Mmjax |
| 023709 | B6.Cg-Hprt1tm364a(CAG-icre/ERT2)Ems/Mmjax |
| 023710 | B6.Cg-Hprt1tm365a(Ple266-icre/ERT2)Ems/Mmjax |
| 023711 | B6.Cg-Hprt1tm366a(Ple67-icre/ERT2)Ems/Mmjax |
| 023712 | B6.Cg-Hprt1tm367a(Ple251-icre/ERT2)Ems/Mmjax |
| 039341 | B6.Cg-Klf1em1(icre)Jbkr/J |
| 038823 | B6.Cg-Myh11em1(icre)Jifz/J |
| 015850 | B6.Cg-Pde6b+ Tg(Rho-icre)1Ck/Boc |
| 008520 | B6.Cg-Tg(CD2-icre)4Kio/J |
| 036935 | B6.FVB(Cg)-Tp(X)1SoffTg(Myh11-icre/ERT2)1Soff/ZjngJ |
| 019079 | B6.FVB-Tg(Myh11-icre/ERT2)1Soff/J |
| 017490 | B6.FVB-Tg(Stra8-icre)1Reb/LguJ |
| 029231 | B6;129-Bhlha15tm4Skz/J |
| 030663 | B6;129S-Gcgtm1.1(icre)Gkg/J |
| 038989 | B6;C3-Oxtrem4(icre/ERT2)Yinn/2J |
| 025809 | B6;CBA-Tg(Fgfr3-icre/ERT2)4-2Wdr/J |
| 026555 | B6;CBA-Tg(Lhx6-icre)1Kess/J |
| 024768 | B6;CBA-Tg(Upk2-icre/ERT2)1Ccc/J |
| 031645 | B6;D2-Tn(pb-CAG-cas9*,-EGFP)1Yangh/J |
| 028547 | B6;SJL-Tg(Cyp17a1-icre)AJako/J |
| 018968 | B6N.Cg-Commd10Tg(Vav1-icre)A2Kio/J |
| 036235 | B6N.Cg-Tg(Cxcl12-icre/ERT2)L289Nono/J |
| 035229 | C57BL/6-Ccr2em1(icre/ERT2)Peng/J |
| 025124 | C57BL/6-Tg(Adipoq-icre/ERT2)1Soff/J |
| 040204 | C57BL/6-Tg(Amelx-icre)872Ppap/JdbJ |
| 038650 | C57BL/6-Tg(Crhr1-icre,-tdTomato)3Njus/J |
| 038197 | C57BL/6-Tg(Nmur1-iCre,-eGFP)1Dart/J |
| 016582 | C57BL/6N-Tg(Slc32a1-icre/ERT2)3Gloss/J |
| 030676 | C57BL/6N-Tg(Tg-cre/ERT2)1Kero/J |
| 021024 | FVB-Tg(Csf1r-icre)1Jwp/J |
| 039016 | NOD.Cg-H2b Commd10Tg(Vav1-icre)A2Kio/JkraJ |
| 008694 | NOD/ShiLt-Tg(Foxp3-EGFP/cre)1cJbs/J |
| 037019 | STOCK Foxp3tm4(icre)Tch/Mmjax |
| 034261 | STOCK Ighg3tm1.1(icre)Gbrt/J |
| 037139 | STOCK Myl1tm1(cre)Sjb Atg7tm1Tchi Gaatm1Rabn/PuroMmjax |
| 039298 | STOCK Shox2em1(icre)Zyliu/J |
| 012691 | STOCK Et(icre/ERT2)14374Rdav/J |
| 012692 | STOCK Et(icre/ERT2)14602Rdav/J |
| 012693 | STOCK Et(icre/ERT2)14624Rdav/J |
| 027798 | STOCK Tg(Nkx6-2-icre)1Kess/SshiJ |
| 018147 | STOCK Tg(Slc17a8-icre)1Edw/SealJ |
| 008208 | STOCK Tg(Stra8-icre)1Reb/J |
| 016584 | STOCK Tg(Tph2-icre/ERT2)6Gloss/J |
| 74 strains use this protocol | |