Genotyping Protocols Database
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 4.0 -- 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
003115 STOCK Tg(CAG-EGFP)B5Nagy/J
003116 STOCK Tg(CAG-EGFP)D4Nagy/J
003291 C57BL/6-Tg(CAG-EGFP)1Osb/J
004190 C.129-Il4tm1Lky/J
004690 B6;FVB-Tg(Pcp2-EGFP)2Yuza/J
005076 NOD.Cg-Tg(tetO-EGFP/FADD)1Doi/DoiJ
005282 NOD/ShiLtJ-Tg(Ins1-EGFP/GH1)14Hara/HaraJ
005418 STOCK Tg(HIST1H2BB/EGFP)1Pa/J
005630 B6.Cg-Tg(Thy1-EYFP)15Jrs/J
005700 C.129P2-Cxcr6tm1Litt/J
005706 C57BL/6-Tg(tetO-CDK5R1/GFP)337Lht/J
005738 B6.FVB-Tg(tetO-EGFP,-Tgfbr2)8Mcle/J
005854 STOCK Tg(Cp-EGFP)25Gaia/J
006043 B6;SJL-Tg(Oxt/EGFP)AI03Wsy/J
006129 STOCK Tg(Zp3-EGFP)1Dean/J
006567 C57BL/6-Tg(CAG-EGFP)131Osb/LeySopJ
006570 STOCK Grm7Tg(SMN2)89Ahmb Smn1tm1Msd Tg(Hlxb9-GFP)1Tmj/J
006614 B6;CBA-Tg(Thy1-CFP/COX8A)C1Lich/J
006617 B6;CBA-Tg(Thy1-CFP/COX8A)S2Lich/J
006664 B6;129P2-Olfr2tm1Mom/MomJ
006665 B6;129P2-Olfr151tm13(Olr226)Mom/MomJ
006850 STOCK Tg(CAG-Bgeo,-NOTCH1,-EGFP)1Lbe/J
007075 CByJ.B6-Tg(CAG-EGFP)1Osb/J
007264 C57BL/6-Tg(Sry-EGFP)92Ei Tg(Sry)4Ei Chr YPOS/EiJ
007673 B6.Cg-Tg(Gad1-EGFP)3Gfng/J
007677 CB6-Tg(Gad1-EGFP)G42Zjh/J
007857 C57BL/6J-Tg(Eno2-YFP/Cox8a)YRwb/J
007860 C57BL/6J-Tg(Eno2-YFP/Cox8a)ZRwb/J
007894 B6.Cg-Tg(Rgs4-EGFP)4Lvt/J
007940 B6.Cg-Tg(Thy1-CFP/COX8A)C1Lich/J
007967 B6.Cg-Tg(Thy1-CFP/COX8A)S2Lich/J
007977 B6;CBA-Tg(Hf/Olfr16-GFP)7Mom/MomJ
007979 B6;CBA-Tg(H/Olfr16-GFP)3Mom/MomJ
007984 B6;CBA-Tg(H/Olfr16-taumCherry,-tauGFP)11Mom/MomJ
007985 B6;CBA-Tg(H/Olfr16-taumCherry,-tauGFP)13Mom/MomJ
007986 B6;CBA-Tg(H*/Olfr16-GFP)11Mom/MomJ
007987 B6;CBA-Tg(H*/Olfr16-GFP)25Mom/MomJ
008126 B6.NOD-Tg(Cd4-EGFP)1Lt/J
008173 NOD.Cg-Tg(Ins1-EGFP)1Hara/QtngJ
009159 B6;FVB-Tg(Cnp-EGFP/Rpl10a)JD368Htz/J
009422 NOD.Cg-Tg(Itgax-Venus)1Mnz/QtngJ
010542 NOD.FVB-Tg(CAG-luc,-GFP)L2G85Chco/FathJ
010713 C57BL/6-Tg(tetO-GFP/tetX)5696Stl/J
011106 STOCK Tg(CAG-GFP*)1Hadj/J
012350 B6;SJL-Tg(Thy1-COP4*H134R/EYFP)20Gfng/J
014548 B6.Cg-Tg(Slc32a1-COP4*H134R/EYFP)8Gfng/J
016925 129;B6-Del(10Grin3b-Tmem259)1Zang Tg(Prnp-Tmem259,-EGFP)6Zhang/J
017466 B6.Cg-Tg(Col1a1*3.6-Topaz)2Rowe/J
017467 C57BL/6-Tg(Dmp1-Topaz)1Ikal/J
017468 STOCK Tg(Col1a1*3.6-Cyan)2Rowe/J
017469 C57BL/6-Tg(BGLAP-Topaz)1Rowe/J
017472 STOCK Tg(Acp5-CFP,Ibsp-YFP,Dmp1-RFP)1Pmay/J
017484 FVB-Tg(JPH3-GFP,-JPH3*)GXwy/J
017919 STOCK Tg(CAG-EGFP,-dsRed2/RNAi:Tardbp)6Zxu/J
017952 STOCK Tg(Isl1-EGFP*)1Slp/J
018071 FVB-Tg(Prism)1861Htz/J
018148 STOCK Tg(Slc17a8-EGFP)1Edw/SealJ
018549 B6.Cg-Tg(Csf1r-EGFP)1Hume/J
018979 B6.129(Cg)-Tg(CAG-EGFP)D4Nagy/KnwJ
019494 B6.Cg-Tg(RP24-131B16/EGFP)37Ghan/J
019908 B6;129S7-Ghsrtm2Rgs/J
021187 FVB(B6)-Tg(Pbsn-rtTA*M2)42Xy/J
021844 129S6.129P2(B6)-Gucy1b2tm2Mom/MomJ
021930 B6.129S1-Tg(CAG-EGFP)S1C2Tpo/KnwPeaJ
021937 NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CAG-EGFP)1Osb/SzJ
021962 B6.129(FVB)-Pitx3tm1.1Cai/J
022086 B6.Cg-Tg(RP24-131B16/EGFP)13Ghan/J
022486 B6.Cg-Ptprca Tg(UBC-PA-GFP)1Mnz/J
023046 B6.129S6(Cg)-Ntrk2tm2.1Ddg/J
023426 STOCK Tg(Kiss1-cre)J2-4Cfe/J
023449 STOCK Myh14tm3.1Rsad/Mmjax
023520 C57BL/6-Bcrtm1(BCR/ABL)Tsr/J
024033 B6.FVB-Tg(Shank3-EGFP)1Hzo/J
024284 STOCK Hcn4tm3Sev/J
024637 B6.129S1(SJL)-Nkx2-5tm2(cre)Rph/J
024750 B6;129S4-Gt(ROSA)26Sortm9(EGFP/Rpl10a)Amc/J
025019 D2.Cg-Tg(Thy1-YFP/Syp)10Jrs/SjJ
025193 STOCK Tg(Vmn1r232-Mapt/EGFP)1Dlc/J
025194 STOCK Tg(Vmn1r206-Mapt/YFP)1Dlc/J
025195 STOCK Tg(Vmn1r191-Mapt/CFP)1Dlc/J
025693 STOCK Tg(CAG-GFP,-mCherry/Pxn)#Mgin/KleyJ
025820 B6;CBA-Tg(CRH-MAPT/Topaz)1Rck/J
025854 B6;FVB-Ptprca Tg(CAG-luc,-GFP)L2G85Chco Thy1a/J
025855 STOCK Ptprca Lag3tm1Doi Tg(CAG-luc,-GFP)L2G85Chco Thy1a/J
026033 STOCK Tg(Aldh1l1-EGFP,-DTA)D8Rth/J
026104 STOCK Tg(Egfl7-EGFP)12Stmn/J
026855 D2.Cg-Tg(Thy1-Brainbow1.1)MLich/SjJ
026856 D2.FVB-Tg(GFAPGFP)14Mes/SjJ
027012 STOCK Foxj1tm1.1(cre/ERT2/GFP)Htg/J
027496 B6.FVB-Tg(Ckm-SLC2A4/EGFP)1Samc/J
027662 C57BL/6-Tyrc-Brd Tg(Gnrhr-luc/EGFP)D8Mrln/J
027665 FVB/N-Tg(Gnrhr-luc/EGFP)L8Mrln/J
027898 C57BL/6J-Tg(CAG-lacZ,-FUS,-EGFP)629Gyu/J
028596 C57BL/6-Tg(Cck-EGFP)2Mirn/J
028623 B6.129P2-S1pr1tm1Hrose/J
028624 B6.Cg-S1pr3tm1.1Hrose/J
029043 B6.Cg-Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze/J
029236 B6.Cg-Pabpn1tm1.1Gpvl/J
029316 C57BL/6-Gt(ROSA)26Sortm1(CAG-FOXO1,GFP)Moli/J
029846 B6.129(Cg)-Rettm13Jmi/SjnJ
029847 B6.129(Cg)-Rettm13.1Jmi/SjnJ
029889 C57BL/6-Tg(CAG-tdTomato,Actb/EGFP)1Erv/J
030252 B6;FVB-Tg(Cort-EGFP/Rpl10a)GM130Htz/J
030255 B6;FVB-Tg(Drd2-EGFP/Rpl10a)CP101Htz/J
030257 B6.FVB(Cg)-Tg(Colgalt2-EGFP/Rpl10a)DU9Htz/J
030265 B6;FVB-Tg(Olig2-EGFP/Rpl10a)JD97Htz/J
030271 B6;FVB-Tg(Sept4-EGFP/Rpl10a)DS152Htz/J
030541 B6.Cg-Foxp2tm1.1(cre)Rpa/J
030544 B6.Cg-Npy1rtm1.1(cre/GFP)Rpa/J
030671 B6.Cg-Tg(Col2a1-EGFP)1Waho/J
031040 STOCK Tg(ACTB-EGFP,-NOX1)1Jdla/J
031279 B6.Cg-Gt(ROSA)26Sortm1(tetO-Yap1*,-GFP*)Stang/J
031621 B6.129S4-Del(14Ltb4r2-Ltb4r1)1Bodd/J
031622 B6.129S4-Ltb4r2tm1Bodd/J
032003 STOCK Grm7Tg(SMN2)89Ahmb Smn1tm1Msd Tg(Hlxb9-GFP)1Tmj Tg(SMN2*delta7)4299Ahmb/J
032886 B6.Cg-Tg(Acta2-GCaMP-ER2)B25-6Mik/J
033064 FVB/N-Tg(ACTA1-ATP2A1*/DsRed/GFP)5Wheel/J
033639 B6;CBA-Tg(Gnrh1-EGFP)51Sumo/J
118 strains use this protocol