Protocol 22102: Probe Assay - Fluorescent Proteins (Generic GFP)
Version 6.0

Notes

Melting curve analysis is done using a Roche Light Cycler 480.

This assay will NOT distinguish hemizygous from homozygous transgenic animals.

This protocol can also be used for YFP.

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 = 92
IPC = 74 bp

 

The Jackson Laboratory designed this protocol using GFP sequence, however, we have found that it can be used to genotype for other fluorescence proteins. It is possible that mismatches in the primer sequences may make data interpretation challenging for other fluorescence proteins.

 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
10201 AGT GCT TCA GCC GCT ACC Transgene Forward A
10202 GAA GAT GGT GCG CTC CTG Transgene Reverse A
13731 Fluorophore-1 TTC AAG TCC GCC ATG CCC GAA Quencher-1 MUT 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
10201 0.40 uM
10202 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
039976 129(Cg)-Gt(ROSA)26Sortm5(CAG-Sun1/sfGFP)Nat/CdnyJ
005483 129-Tg(CAG-EYFP)7AC5Nagy/J
034473 129;B6-Otx2tm2(cre/ERT2)Tlam/J
016925 129;B6-Del(10Grin3b-Tmem259)1Zang Tg(Prnp-Tmem259,-EGFP)6Zhang/J
021844 129S6.129P2(B6)-Gucy1b2tm2Mom/MomJ
032051 B6(129S4)-Kittm1.1(cre)Jmol/J
038534 B6(Cg)-Foxp3tm1.1(flpo)Daav/J
039195 B6(Cg)-Il17atm1.1(EYFP/icre)Ehs/J
039963 B6(Cg)-Tyrc-2J H2-Eb1em1Jmlr/JmlrJ
038536 B6(Cg)-Tg(Cd8a-icre/ERT2,-GFP)#Daav/J
033943 B6.129(C)-Bdnftm1.1Bail/KmrtJ
035371 B6.129(Cg)-Msi2tm1.1Geno/ReyaJ
029846 B6.129(Cg)-Rettm13Jmi/SjnJ
004178 B6.129(Cg)-Tg(CAG-Bgeo/GFP)21Lbe/J
018979 B6.129(Cg)-Tg(CAG-EGFP)D4Nagy/KnwJ
030420 B6.129(FVB)-Gt(ROSA)26Sortm1(CAG-birA,-EGFP)Joez/J
004218 B6.129(ICR)-Tg(CAG-ECFP)CK6Nagy/J
039728 B6.129-Gt(ROSA)26Sortm1(CAG-Sall4_ia,-Sall4_ib,-EGFP)Ykawa/J
030952 B6.129-Gt(ROSA)26Sortm5.1(CAG-Sun1/sfGFP)Nat/MmbeJ
032536 B6.129P2-Aviltm2(cre)Fawa/J
028623 B6.129P2-S1pr1tm1Hrose/J
021930 B6.129S1-Tg(CAG-EGFP)S1C2Tpo/KnwPeaJ
031622 B6.129S4-Ltb4r2tm1Bodd/J
031621 B6.129S4-Del(14Ltb4r2-Ltb4r1)1Bodd/J
023046 B6.129S6(Cg)-Ntrk2tm2.1Ddg/J
012904 B6.129S6-S100a4tm1Egn/YunkJ
036498 B6.Cg-Airetm2Mmat/EhrlJ
033942 B6.Cg-Bdnftm2.1Kmrt/J
039897 B6.Cg-Ccl3tm1.1(HBEGF)Ckur/J
030305 B6.Cg-Eef1a1tm1Rck/J
030541 B6.Cg-Foxp2tm1.1(cre)Rpa/J
024953 B6.Cg-Giptm1.1Gmost/J
031279 B6.Cg-Gt(ROSA)26Sortm1(tetO-Yap1*,-GFP*)Stang/J
040817 B6.Cg-Gt(ROSA)26Sortm16(CAG-RRAS2,-EGFP)Geno/J
026944 B6.Cg-Gt(ROSA)26Sortm2.1(CAG-EGFP,-DTA*G128D)Pjen/J
029043 B6.Cg-Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze/J
037379 B6.Cg-Igs7tm211(tetO-ChrimsonR/tdTomato,CAG-tTA2)Tasic/J
037380 B6.Cg-Igs7tm214(CAG-ACR1*,CAG-mRuby3)Tasic/J
030544 B6.Cg-Npy1rtm1.1(cre/GFP)Rpa/J
029236 B6.Cg-Pabpn1tm1.1Gpvl/J
040737 B6.Cg-Ptprca Pepcb Gt(ROSA)26Sortm1.1(CAG-cas9*,-EGFP)Fezh Tg(TcraTcrb)425Cbn/ShrJ
022486 B6.Cg-Ptprca Tg(UBC-PA-GFP)1Mnz/J
028624 B6.Cg-S1pr3tm1.1Hrose/J
039726 B6.Cg-Sall4tm1.1(EGFP/cre/ERT2)Ykawa/J
038835 B6.Cg-Sdk1em1(cre/GFP)Jrs/KrishJ
017466 B6.Cg-Tg(Col1a1*3.6-Topaz)2Rowe/J
030671 B6.Cg-Tg(Col2a1-EGFP)1Waho/J
018549 B6.Cg-Tg(Csf1r-EGFP)1Hume/J
018306 B6.Cg-Tg(Fos-tTA,Fos-EGFP*)1Mmay/J
014135 B6.Cg-Tg(Fos/EGFP)1-3Brth/J
007673 B6.Cg-Tg(Gad1-EGFP)3Gfng/J
006069 B6.Cg-Tg(HIST1H2BB/EGFP)1Pa/J
005029 B6.Cg-Tg(Hlxb9-GFP)1Tmj/J
006864 B6.Cg-Tg(Ins1-EGFP)1Hara/J
012643 B6.Cg-Tg(Ly6a-EGFP)G5Dzk/J
007894 B6.Cg-Tg(Rgs4-EGFP)4Lvt/J
022086 B6.Cg-Tg(RP24-131B16/EGFP)13Ghan/J
019494 B6.Cg-Tg(RP24-131B16/EGFP)37Ghan/J
007940 B6.Cg-Tg(Thy1-CFP/COX8A)C1Lich/J
007967 B6.Cg-Tg(Thy1-CFP/COX8A)S2Lich/J
007615 B6.Cg-Tg(Thy1-COP4/EYFP)9Gfng/J
005630 B6.Cg-Tg(Thy1-EYFP)15Jrs/J
030257 B6.FVB(Cg)-Tg(Colgalt2-EGFP/Rpl10a)DU9Htz/J
018056 B6.FVB-Tg(CAG-boNT/B,-EGFP)U75-56Fwp/J
027496 B6.FVB-Tg(Ckm-SLC2A4/EGFP)1Samc/J
006000 B6.FVB-Tg(ITGAM-HBEGF/EGFP)34Lan/J
005738 B6.FVB-Tg(tetO-EGFP,-Tgfbr2)8Mcle/J
008126 B6.NOD-Tg(Cd4-EGFP)1Lt/J
021039 B6;129-Gt(ROSA)26Sortm5(CAG-Sun1/sfGFP)Nat/J
021162 B6;129P2-Mapttm2Arbr/J
006664 B6;129P2-Or6a2tm1Mom/MomJ
006665 B6;129P2-Or8a1tm13(Olr226)Mom/MomJ
028581 B6;129S4-Gt(ROSA)26Sortm2(CAG-HIST1H2BB/EGFP)Zjh/J
024750 B6;129S4-Gt(ROSA)26Sortm9(EGFP/Rpl10a)Amc/J
038118 B6;129S6-Ank3tm1Pmj/J
017495 B6;129S7-Crim1tm1(GFP/cre/ERT2)Pzg/J
030537 B6;129S7-Drd2tm1.1Jtw/J
019908 B6;129S7-Ghsrtm2Rgs/J
034237 B6;129X-Stx2tm1Dcru/J
039470 B6;129X1-Nfe2l1tm1Jefc/J
025820 B6;CBA-Tg(CRH-MAPT/Topaz)1Rck/J
033639 B6;CBA-Tg(Gnrh1-EGFP)51Sumo/J
007986 B6;CBA-Tg(H*/Olfr16-GFP)11Mom/MomJ
007987 B6;CBA-Tg(H*/Olfr16-GFP)25Mom/MomJ
007979 B6;CBA-Tg(H/Olfr16-GFP)3Mom/MomJ
007980 B6;CBA-Tg(H/Olfr16-GFP)4Mom/MomJ
007984 B6;CBA-Tg(H/Olfr16-taumCherry,-tauGFP)11Mom/MomJ
007985 B6;CBA-Tg(H/Olfr16-taumCherry,-tauGFP)13Mom/MomJ
007977 B6;CBA-Tg(Hf/Olfr16-GFP)7Mom/MomJ
006614 B6;CBA-Tg(Thy1-CFP/COX8A)C1Lich/J
006617 B6;CBA-Tg(Thy1-CFP/COX8A)S2Lich/J
031645 B6;D2-Tn(pb-CAG-dcas9,-EGFP)1Yangh/J
038774 B6;D2-Tg(PDGFB-SNCA/EGFP)78Ema/RoriJ
005621 B6;D2-Tg(S100B-EGFP)1Wjt/J
008344 B6;DBA-Tg(Fos-tTA,Fos-EGFP*)1Mmay Tg(tetO-lacZ,tTA*)1Mmay/J
025854 B6;FVB-Ptprca Tg(CAG-luc,-GFP)L2G85Chco Thy1a/J
009159 B6;FVB-Tg(Cnp-EGFP/Rpl10a)JD368Htz/J
030252 B6;FVB-Tg(Cort-EGFP/Rpl10a)GM130Htz/J
037194 B6;FVB-Tg(Cx3cl1-EGFP/Rpl10)ES26Htz/J
030255 B6;FVB-Tg(Drd2-EGFP/Rpl10a)CP101Htz/J
037198 B6;FVB-Tg(Gdnf-EGFP/Rpl10)ES2243Htz/J
030265 B6;FVB-Tg(Olig2-EGFP/Rpl10a)JD97Htz/J
004690 B6;FVB-Tg(Pcp2-EGFP)2Yuza/J
030271 B6;FVB-Tg(Sept4-EGFP/Rpl10a)DS152Htz/J
006043 B6;SJL-Tg(Oxt/EGFP)AI03Wsy/J
012350 B6;SJL-Tg(Thy1-COP4*H134R/EYFP)20Gfng/J
040426 B6J(B6N)-Tg(Nr4a1-EGFP/cre)820Khog/PalcaJ
035474 B6N.Cg-Tg(Arc-tTA,-EGFP/Capzb*)10Mkyph/J
004190 C.129-Il4tm1Lky/J
005700 C.129P2-Cxcr6tm1Litt/J
040918 C57BL/6-Ahrtm1Lgzh/J
023520 C57BL/6-Bcrtm1(BCR/ABL)Tsr/J
038343 C57BL/6-Cnr2tm1.1Geno/J
037626 C57BL/6-Gt(ROSA)26Sorem1(CAG-Kcnq2*/EGFP)Hjch/J
029316 C57BL/6-Gt(ROSA)26Sortm1(CAG-FOXO1,GFP)Moli/J
039713 C57BL/6-Pgbd5tm2.1Aken/J
039454 C57BL/6-Proser2em3Odk/Mmjax
036868 C57BL/6-Rnf111em1Litt/J
040504 C57BL/6-Sting1em3Nyan/J
027662 C57BL/6-Tyrc-Brd Tg(Gh1-luc/EGFP)D8Mrln/J
017469 C57BL/6-Tg(BGLAP-Topaz)1Rowe/J
006567 C57BL/6-Tg(CAG-EGFP)131Osb/LeySopJ
003291 C57BL/6-Tg(CAG-EGFP)1Osb/J
029889 C57BL/6-Tg(CAG-tdTomato,Actb/EGFP)1Erv/J
028596 C57BL/6-Tg(Cck-EGFP)2Mirn/J
005070 C57BL/6-Tg(Csf1r-EGFP-NGFR/FKBP1A/TNFRSF6)2Bck/J
017467 C57BL/6-Tg(Dmp1-Topaz)1Ikal/J
016617 C57BL/6-Tg(Nr4a1-EGFP/cre)820Khog/J
032131 C57BL/6-Tg(Scx-GFP/cre)1Stzr/J
007264 C57BL/6-Tg(Sry-EGFP)92Ei Tg(Sry)4Ei Chr YPOS/EiJ
005706 C57BL/6-Tg(tetO-CDK5R1/GFP)337Lht/J
010713 C57BL/6-Tg(tetO-GFP/tetX)5696Stl/J
004353 C57BL/6-Tg(UBC-GFP)30Scha/J
040457 C57BL/6J-Gprc5aem1(icre/ERT2)Blr/Mmjax
040505 C57BL/6J-Gt(ROSA)26Sorem1(CAG-Sting1,-EGFP)Nyan/J
040416 C57BL/6J-Igs2em1(CAG-mCherry,-Cxcr3*,-GFP*)Miji/J
040208 C57BL/6J-Igs2em1(CAG-RFP*/GFP*)Qsun/J
036197 C57BL/6J-Nr1i2em1Neg/J
041309 C57BL/6J-Tcf19em1Jsun/J
038605 C57BL/6J-Tg(CAG-GFP*,-ATP6AP2)1Vli/J
027898 C57BL/6J-Tg(CAG-lacZ,-FUS,-EGFP)629Gyu/J
006362 C57BL/6J-Tg(CMV-Cox8a/EYFP)17J/J
040140 C57BL/6J-Tg(Cxcl9-RFP,Cxcl10-BFP)1Adl/J
007857 C57BL/6J-Tg(Eno2-YFP/Cox8a)YRwb/J
007860 C57BL/6J-Tg(Eno2-YFP/Cox8a)ZRwb/J
009593 C57BL/6J-Tg(Pomc-EGFP)1Low/J
039123 C57BL/6N-Tracem1Gmrs/J
028324 C57BL/6NJ-Gpr15em2J/J
007677 CB6-Tg(Gad1-EGFP)G42Zjh/J
007075 CByJ.B6-Tg(CAG-EGFP)1Osb/J
007076 CByJ.B6-Tg(UBC-GFP)30Scha H2b/J
026855 D2.Cg-Tg(Thy1-Brainbow1.1)MLich/SjJ
025019 D2.Cg-Tg(Thy1-YFP/Syp)10Jrs/SjJ
026856 D2.FVB-Tg(GFAPGFP)14Mes/SjJ
021187 FVB(B6)-Tg(Pbsn-rtTA*M2)42Xy/J
003718 FVB-Tg(GadGFP)45704Swn/J
005515 FVB-Tg(ITGAM-HBEGF/EGFP)34Lan/J
017484 FVB-Tg(JPH3-GFP,-JPH3*)GXwy/J
040892 FVB-Tg(NPHS2-EGFP)#Pne/LucaiJ
018071 FVB-Tg(Prism)1861Htz/J
003516 FVB.Cg-Tg(CAG-EGFP)B5Nagy/J
033064 FVB/N-Tg(ACTA1-ATP2A1*/DsRed/GFP)5Wheel/J
028698 FVB/N-Tg(CAG-EGFP,-Tnf)1Kul/J
003257 FVB/N-Tg(GFAPGFP)14Mes/J
027665 FVB/N-Tg(Gnrhr-luc/EGFP)L8Mrln/J
040440 NOD.B6(Cg)-Tg(Ins1-INS/sfGFP)369Arvn/J
021937 NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CAG-EGFP)1Osb/SzJ
008173 NOD.Cg-Tg(Ins1-EGFP)1Hara/QtngJ
009422 NOD.Cg-Tg(Itgax-Venus)1Mnz/QtngJ
005076 NOD.Cg-Tg(tetO-EGFP/FADD)1Doi/DoiJ
010542 NOD.FVB-Tg(CAG-luc,-GFP)L2G85Chco/FathJ
008547 NOD.FVB-Tg(ITGAM-HBEGF/EGFP)34Lan/JdkJ
008694 NOD/ShiLt-Tg(Foxp3-EGFP/cre)1cJbs/J
005282 NOD/ShiLtJ-Tg(Ins1-EGFP/GH1)14Hara/HaraJ
036735 STOCK Adgra3tm1.1(HBEGF,-cre/ERT2)Pac/Mmjax
012881 STOCK Ascl1tm1Reed/J
040665 STOCK Cntn2tm1(DTA)Dkar/J
027012 STOCK Foxj1tm1.1(cre/ERT2/GFP)Htg/J
040090 STOCK Gli1tm4.1Alj/J
032003 STOCK Grm7Tg(SMN2)89Ahmb Smn1tm1Msd Tg(Hlxb9-GFP)1Tmj Tg(SMN2*delta7)4299Ahmb/J
006570 STOCK Grm7Tg(SMN2)89Ahmb Smn1tm1Msd Tg(Hlxb9-GFP)1Tmj/J
038583 STOCK Gt(ROSA)26Sorem1(CAG-Atoh1/EGFP)Akg/J
038584 STOCK Gt(ROSA)26Sorem2(CAG-Gf1i,-Atoh1/EGFP)Akg/J
038585 STOCK Gt(ROSA)26Sorem3(CAG-Gf1i,-Atoh1/EGFP,-Pou4f3)Akg/J
039898 STOCK Gt(ROSA)26Sortm1(CAG-Atf4/EGFP,-tdTomato)Calak/J
039717 STOCK Gt(ROSA)26Sortm16(CAG-Nsd2,-EBFP)Rsky/J
040009 STOCK Gt(ROSA)26Sortm3(tTA,tetO-TREM2,-EGFP)Gbu/NzhaoJ
040010 STOCK Gt(ROSA)26Sortm4(tTA,tetO-TREM2*R47H,-EGFP)Gbu/NzhaoJ
024284 STOCK Hcn4tm3Sev/J
040818 STOCK Hoxa5em1Dmwe/J
040717 STOCK Igs2em1(HNF4A-Gsta2,-H2B/EGFP)Amc/J
034694 STOCK Igs7tm86(tetO-CiVS/pHluorin)Hze/VpiJ
023449 STOCK Myh14tm3.1Rsad/Mmjax
039204 STOCK Neurog2tm1Cschu/J
039935 STOCK Piezo2em1.1Ddg/J
039838 STOCK Ptentm1.1Ayr/J
025855 STOCK Ptprca Lag3tm1Doi Tg(CAG-luc,-GFP)L2G85Chco Thy1a/J
036906 STOCK Tmem178btm1c(KOMP)Wtsi Tmem178tm1.1Sud/J
040657 STOCK Tmprss2tm1.1(cre/ERT2)Ychen/J
039724 STOCK Vimtm2(flpo)Ggen/J
010911 STOCK Wt1tm1(EGFP/cre)Wtp/J
039156 STOCK Zic5tm1.1(icre/ERT2)Akg/J
017472 STOCK Tg(Acp5-CFP,Ibsp-YFP,Dmp1-RFP)1Pmay/J
026033 STOCK Tg(Aldh1l1-EGFP,-DTA)D8Rth/J
006850 STOCK Tg(CAG-Bgeo,-NOTCH1,-EGFP)1Lbe/J
003920 STOCK Tg(CAG-Bgeo/GFP)21Lbe/J
003115 STOCK Tg(CAG-EGFP)B5Nagy/J
003116 STOCK Tg(CAG-EGFP)D4Nagy/J
017919 STOCK Tg(CAG-EGFP,-dsRed2/RNAi:Tardbp)6Zxu/J
038303 STOCK Tg(CAG-EGFP/Map1lc3b)53Nmz Gaatm1Rabn/RabnMmjax
011106 STOCK Tg(CAG-GFP*)1Hadj/J
017468 STOCK Tg(Col1a1*3.6-Cyan)2Rowe/J
005854 STOCK Tg(Cp-EGFP)25Gaia/J
018322 STOCK Tg(Cp-EGFP)25Gaia/ReyaJ
026104 STOCK Tg(Egfl7-EGFP)12Stmn/J
039609 STOCK Tg(HBE1-H2B/CFP)30Maba/J
039607 STOCK Tg(HBE1-H2B/EGFP)55Maba/J
005418 STOCK Tg(HIST1H2BB/EGFP)1Pa/J
017952 STOCK Tg(Isl1-EGFP*)1Slp/J
039608 STOCK Tg(Kdr-H2B/EYFP)32Maba/J
018148 STOCK Tg(Slc17a8-EGFP)1Edw/SealJ
012345 STOCK Tg(tetO-tdTomato,-Syp/EGFP*)1.1Luo/J
025195 STOCK Tg(Vmn1r191-Mapt/CFP)1Dlc/J
025194 STOCK Tg(Vmn1r206-Mapt/YFP)1Dlc/J
025193 STOCK Tg(Vmn1r232-Mapt/EGFP)1Dlc/J
006129 STOCK Tg(Zp3-EGFP)1Dean/J
226 strains use this protocol