Protocol 22182: QPCR Assay - Generic GFP/EGFP qPCR

B6.129(ICR)-Tg(CAG-ECFP)CK6Nagy/J

Stock No: 004218

Version 3.0

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
14977		AAG GGC ATC GAC TTC AAG G		Transgene Forward	A
14978		TGC TTG TCG GCC ATG ATA TAG		Transgene Reverse	A
14979	Fluorophore-1	CTT GTG CCC CAG GAT GTT GCC	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
14977	0.40 uM
14978	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
034473	129;B6-Otx2^tm2(cre/ERT2)Tlam/J
003960	129S6-Tg(Prnp-GFP/cre)1Blw/J
032051	B6(129S4)-Kit^tm1.1(cre)Jmol/J
033943	B6.129(C)-Bdnf^tm1.1Bail/KmrtJ
035371	B6.129(Cg)-Msi2^tm1.1Geno/ReyaJ
030420	B6.129(FVB)-Gt(ROSA)26Sor^tm1(CAG-birA,-EGFP)Joez/J
004218	B6.129(ICR)-Tg(CAG-ECFP)CK6Nagy/J
030952	B6.129-Gt(ROSA)26Sor^tm5.1(CAG-Sun1/sfGFP)Nat/MmbeJ
032536	B6.129P2-Avil^tm2(cre)Fawa/J
033942	B6.Cg-Bdnf^tm2.1Kmrt/J
030305	B6.Cg-Eef1a1^tm1Rck/J
024953	B6.Cg-Gip^tm1.1Gmost/J
031279	B6.Cg-Gt(ROSA)26Sor^tm1(tetO-Yap1*,-GFP*)Stang/J
026944	B6.Cg-Gt(ROSA)26Sor^tm2.1(CAG-EGFP,-DTA*G128D)Pjen/J
037379	B6.Cg-Igs7^tm211(tetO-ChrimsonR/tdTomato,CAG-tTA2)Tasic/J
037380	B6.Cg-Igs7^tm214(CAG-ACR1*,CAG-mRuby3)Tasic/J
028719	B6.Cg-Relb^tm1Ukl/J
005884	B6.Cg-Tg(CAG-mRFP1)1F1Hadj/J
030671	B6.Cg-Tg(Col2a1-EGFP)1Waho/J
018549	B6.Cg-Tg(Csf1r-EGFP)1Hume/J
014135	B6.Cg-Tg(Fos/EGFP)1-3Brth/J
006864	B6.Cg-Tg(Ins1-EGFP)1Hara/J
037887	B6.Cg-Tg(Ins1-Mir30d,-EGFP)HXtng/J
037886	B6.Cg-Tg(Ins1-Mir30d,-EGFP)LXtng/J
031696	B6.Cg-Tg(Itgax-FCER1A,-EGFP)1Maur/J
036759	B6.Cg-Tg(Pax7-EGFP)15Tajb/J
007902	B6.Cg-Tg(RP23-268L19-EGFP)2Mik/J
005630	B6.Cg-Tg(Thy1-EYFP)15Jrs/J
018056	B6.FVB-Tg(CAG-boNT/B,-EGFP)U75-56Fwp/J
018055	B6.FVB-Tg(H2-K-S100a9,GFP)1Gabr/J
021039	B6;129-Gt(ROSA)26Sor^tm5(CAG-Sun1/sfGFP)Nat/J
017495	B6;129S7-Crim1^tm1(GFP/cre/ERT2)Pzg/J
030537	B6;129S7-Drd2^tm1.1Jtw/J
034237	B6;129X-Stx2^tm1Dcru/J
025820	B6;CBA-Tg(CRH-MAPT/Topaz)1Rck/J
033639	B6;CBA-Tg(Gnrh1-EGFP)51Sumo/J
033357	B6;CBA-Tg(Plp1-EGFP)10Wmac/J
038774	B6;D2-Tg(PDGFB-SNCA/EGFP)78Ema/RoriJ
031645	B6;D2-Tn(pb-CAG-cas9*,-EGFP)1Yangh/J
008344	B6;DBA-Tg(Fos-tTA,Fos-EGFP*)1Mmay Tg(tetO-lacZ,tTA*)1Mmay/J
025854	B6;FVB-Ptprc^a Tg(CAG-luc,-GFP)L2G85Chco Thy1^a/J
037194	B6;FVB-Tg(Cx3cl1-EGFP/Rpl10)ES26Htz/J
037198	B6;FVB-Tg(Gdnf-EGFP/Rpl10)ES2243Htz/J
028356	B6N.Cg-Tg(Sftpc,-EGFP)1Dobb/J
004190	C.129-Il4^tm1Lky/J
038343	C57BL/6-Cnr2^tm1.1Geno/J
019108	C57BL/6-Efna3^tm1Ebp/J
037626	C57BL/6-Gt(ROSA)26Sor^em1(CAG-Kcnq2*/EGFP)Hjch/J
036868	C57BL/6-Rnf111^em1Litt/J
027662	C57BL/6-Tyr^c-Brd Tg(Gh1-luc/EGFP)D8Mrln/J
006567	C57BL/6-Tg(CAG-EGFP)131Osb/LeySopJ
005070	C57BL/6-Tg(Csf1r-EGFP-NGFR/FKBP1A/TNFRSF6)2Bck/J
037185	C57BL/6-Tg(SV40-Sox11,-EGFP)2Spare/J
036197	C57BL/6J-Nr1i2^em1Neg/J
007857	C57BL/6J-Tg(Eno2-YFP/Cox8a)YRwb/J
007677	CB6-Tg(Gad1-EGFP)G42Zjh/J
007076	CByJ.B6-Tg(UBC-GFP)30Scha H2^b/J
008450	FVB-Tg(CAG-luc,-GFP)L2G85Chco/J
003718	FVB-Tg(GadGFP)45704Swn/J
003516	FVB.Cg-Tg(CAG-EGFP)B5Nagy/J
028698	FVB/N-Tg(CAG-EGFP,-Tnf)1Kul/J
025062	FVB/N-Tg(Figla-EGFP,-icre)ZP3Dean/Mmjax
023410	FVB/N-Tg(tetO-Nog,-EGFP)53Hda/J
010542	NOD.FVB-Tg(CAG-luc,-GFP)L2G85Chco/FathJ
036735	STOCK Adgra3^tm1.1(HBEGF,-cre/ERT2)Pac/Mmjax
036496	STOCK Aire^tm4.1(AIRE/GFP)Mmat/J
032003	STOCK Grm7^Tg(SMN2)89Ahmb Smn1^tm1Msd Tg(Hlxb9-GFP)1Tmj Tg(SMN2*delta7)4299Ahmb/J
038583	STOCK Gt(ROSA)26Sor^em1(CAG-Atoh1/EGFP)Akg/J
038584	STOCK Gt(ROSA)26Sor^em2(CAG-Gf1i,-Atoh1/EGFP)Akg/J
038585	STOCK Gt(ROSA)26Sor^em3(CAG-Gf1i,-Atoh1/EGFP,-Pou4f3)Akg/J
017530	STOCK Igs2^tm2(ACTB-tdTomato,-EGFP)Luo Trp53^tm1Tyj Nf1^tm1Par/J
013751	STOCK Igs2^tm2(ACTB-tdTomato,-EGFP)Luo/J
034694	STOCK Igs7^tm86(tetO-CiVS/pHluorin)Hze/VpiJ
036906	STOCK Tmem178b^tm1c(KOMP)Wtsi Tmem178^tm1.1Sud/J
010911	STOCK Wt1^tm1(EGFP/cre)Wtp/J
003116	STOCK Tg(CAG-EGFP)D4Nagy/J
025693	STOCK Tg(CAG-GFP,-mCherry/Pxn)#Mgin/KleyJ
018322	STOCK Tg(Cp-EGFP)25Gaia/ReyaJ
023345	STOCK Tg(Pgk1-Ccnb1/EGFP)1Aklo/J
012345	STOCK Tg(tetO-tdTomato,-Syp/EGFP*)1.1Luo/J
80 strains use this protocol