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Melting curve analysis is done using a Roche Light Cycler 480.
This assay will NOT distinguish hemizygous from homozygous transgenic animals.
Transgene = 236 bp
Internal positive control = 324 bp
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
oIMR7338 | CTA GGC CAC AGA ATT GAA AGA TCT | Internal Positive Control Forward | A | |||
oIMR7339 | GTA GGT GGA AAT TCT AGC ATC ATC C | Internal Positive Control Reverse | A | |||
oIMR9266 | AGA TGC CAG GAC ATC AGG AAC CTG | Transgene Forward | A | iCre F | ||
oIMR9267 | ATC AGC CAC ACC AGA CAC AGA GAT C | Transgene Reverse | A | iCre R |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTP KAPA | 0.26 mM |
oIMR7338 | 0.50 uM |
oIMR7339 | 0.50 uM |
oIMR9266 | 0.50 uM |
oIMR9267 | 0.50 uM |
Glycerol | 6.50 % |
Dye | 1.00 X |
Kapa 2G HS taq polym | 0.03 U/ul |
DNA |
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 |
Stock Number | Strain Name |
---|---|
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 |
008610 | B6.Cg-Commd10Tg(Vav1-icre)A2Kio/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 |
008520 | B6.Cg-Tg(CD2-icre)4Kio/J |
036935 | B6.FVB(Cg)-Tp(X)1SoffTg(Myh11-icre/ERT2)1Soff/ZjngJ |
017490 | B6.FVB-Tg(Stra8-icre)1Reb/LguJ |
029231 | B6;129-Bhlha15tm4Skz/J |
030663 | B6;129S-Gcgtm1.1(icre)Gkg/J |
025809 | B6;CBA-Tg(Fgfr3-icre/ERT2)4-2Wdr/J |
026555 | B6;CBA-Tg(Lhx6-icre)1Kess/J |
018968 | B6N.Cg-Commd10Tg(Vav1-icre)A2Kio/J |
030676 | C57BL/6N-Tg(Tg-cre/ERT2)1Kero/J |
021024 | FVB-Tg(Csf1r-icre)1Jwp/J |
008694 | NOD/ShiLt-Tg(Foxp3-EGFP/cre)1cJbs/J |
034261 | STOCK Ighg3tm1.1(icre)Gbrt/J |
037139 | STOCK Myl1tm1(cre)Sjb Atg7tm1Tchi Gaatm1Rabn/PuroMmjax |
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 |
49 strains use this protocol |