Protocol 22178: QPCR Assay - iCre QPCR

C57BL/6N-Tg(Tg-cre/ERT2)1Kero/J

Stock No: 030676

Version 4.0

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination. The transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.

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

JAX Protocol

Protocol Primers

Primer	5' Label	Sequence 5' → 3'	3' Label	Primer Type	Reaction	Note
17817		ACA ACT ACC TGT TCT GCC G		Forward	A
17818		GCC TCA AAG ATC CCT TCC AG		Reverse	A
17819	Fluorophore-1	TTC TGC CAC CTC CCA ACT GTC C	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
ddH2O
Kapa Probe Fast QPCR	1.00 X
17817	0.40 uM
17818	0.40 uM
oIMR1544	0.40 uM
oIMR3580	0.40 uM
Tg Probe	0.15 uM
IC Probe	0.15 uM
DNA

Cycling

Step	Temp °C	Time	Note
1	95.0	--
2	95.0	--
3	60.0	--	repeat steps 2-3 for 40 cycles

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)-Jchain^em1(icre/ERT2)Deep/J
035435	B6(129S4)-Xcr1^tm1.1(cre)Kmm/J
010696	B6(129S4)-Et(icre/ERT2)10596Rdav/J
010697	B6(129S4)-Et(icre/ERT2)10727Rdav/J
037836	B6(FVB)-Il1b^em1Mora/J
038129	B6.Cg-Ascl3^tm2.1(EGFP/icre/ERT2)Ovi/J
038342	B6.Cg-Gas1^em1(icre/ERT2)Nono/J
023691	B6.Cg-Hprt1^tm346a(Ple264-icre/ERT2)Ems/Mmjax
026516	B6.Cg-Tg(Avil-icre/ERT2)AJwo/J
008520	B6.Cg-Tg(CD2-icre)4Kio/J
036935	B6.FVB(Cg)-Tp(X)1Soff^Tg(Myh11-icre/ERT2)1Soff/ZjngJ
019079	B6.FVB-Tg(Myh11-icre/ERT2)1Soff/J
017490	B6.FVB-Tg(Stra8-icre)1Reb/LguJ
030663	B6;129S-Gcg^tm1.1(icre)Gkg/J
025809	B6;CBA-Tg(Fgfr3-icre/ERT2)4-2Wdr/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
036235	B6N.Cg-Tg(Cxcl12-icre/ERT2)L289Nono/J
035229	C57BL/6-Ccr2^em1(icre/ERT2)Peng/J
025124	C57BL/6-Tg(Adipoq-icre/ERT2)1Soff/J
038197	C57BL/6-Tg(Nmur1-iCre,-eGFP)1Dart/J
016583	C57BL/6-Tg(Slc6a3-icre/ERT2)2Gloss/J
030676	C57BL/6N-Tg(Tg-cre/ERT2)1Kero/J
019098	FVB-Tg(Csf1r-cre/Esr1*)1Jwp/J
021024	FVB-Tg(Csf1r-icre)1Jwp/J
037019	STOCK Foxp3^tm4(icre)Tch/Mmjax
034261	STOCK Ighg3^tm1.1(icre)Gbrt/J
037139	STOCK Myl1^tm1(cre)Sjb Atg7^tm1Tchi Gaa^tm1Rabn/PuroMmjax
008208	STOCK Tg(Stra8-icre)1Reb/J
030061	STOCK Tg(Tbx18-icre)3Fech/J
016584	STOCK Tg(Tph2-icre/ERT2)6Gloss/J
32 strains use this protocol