Protocol 29498: Standard PCR Assay - Fluorescent Proteins (Generic GFP)
Version 4.2

Notes

This assay will NOT distinguish hemizygous from homozygous transgenic animals.
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 = 173 bp
Internal positive control = 324 bp
Separated by gel electrophoresis on a 1.5% agarose gel.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR0872 AAG TTC ATC TGC ACC ACC G Transgene A GFP
oIMR1416 TCC TTG AAG AAG ATG GTG CG Transgene A GFP
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

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
oIMR0872 0.50 uM
oIMR1416 0.50 uM
oIMR7338 0.50 uM
oIMR7339 0.50 uM
Glycerol 6.50 %
Dye 1.00 X
Kapa 2G HS taq polym 0.03 U/ul
DNA

Cycling

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
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
JAX uses a 'touchdown' cycling protocol and therefore has not calculated the optimal annealing temperature for each set of primers.

Strains Using This Protocol

Stock Number Strain Name
005483 129-Tg(CAG-EYFP)7AC5Nagy/J
004178 B6.129(Cg)-Tg(CAG-Bgeo/GFP)21Lbe/J
004218 B6.129(ICR)-Tg(CAG-ECFP)CK6Nagy/J
017466 B6.Cg-Tg(Col1a1*3.6-Topaz)2Rowe/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
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
005738 B6.FVB-Tg(tetO-EGFP,-Tgfbr2)8Mcle/J
008126 B6.NOD-Tg(Cd4-EGFP)1Lt/J
006665 B6;129P2-Olfr151tm13(Olr226)Mom/MomJ
017495 B6;129S7-Crim1tm1(GFP/cre/ERT2)Pzg/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
008344 B6;DBA-Tg(Fos-tTA,Fos-EGFP*)1Mmay Tg(tetO-lacZ,tTA*)1Mmay/J
009159 B6;FVB-Tg(Cnp-EGFP/Rpl10a)JD368Htz/J
004690 B6;FVB-Tg(Pcp2-EGFP)2Yuza/J
006043 B6;SJL-Tg(Oxt/EGFP)AI03Wsy/J
012350 B6;SJL-Tg(Thy1-COP4*H134R/EYFP)20Gfng/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
005070 C57BL/6-Tg(Csf1r-EGFP-NGFR/FKBP1A/TNFRSF6)2Bck/J
017467 C57BL/6-Tg(Dmp1-Topaz)1Ikal/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
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
007075 CByJ.B6-Tg(CAG-EGFP)1Osb/J
007076 CByJ.B6-Tg(UBC-GFP)30Scha H2b/J
003718 FVB-Tg(GadGFP)45704Swn/J
005515 FVB-Tg(ITGAM-DTR/EGFP)34Lan/J
017484 FVB-Tg(JPH3-GFP,-JPH3*)GXwy/J
018071 FVB-Tg(Prism)1861Htz/J
003257 FVB/N-Tg(GFAPGFP)14Mes/J
013233 NOD.B6-Tg(Itgax-cre,-EGFP)4097Ach/J
010542 NOD.FVB-Tg(CAG-luc,-GFP)L2G85Chco/FathJ
008547 NOD.FVB-Tg(ITGAM-DTR/EGFP)34Lan/JdkJ
008694 NOD/ShiLt-Tg(Foxp3-EGFP/cre)1cJbs/J
005282 NOD/ShiLtJ-Tg(Ins1-EGFP/GH1)14Hara/HaraJ
006570 STOCK Grm7Tg(SMN2)89Ahmb Smn1tm1Msd Tg(Hlxb9-GFP)1Tmj/J
017472 STOCK Tg(Acp5-CFP,Ibsp-YFP,Dmp1-RFP)1Pmay/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
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
005418 STOCK Tg(HIST1H2BB/EGFP)1Pa/J
017952 STOCK Tg(Isl1-EGFP*)1Slp/J
006129 STOCK Tg(Zp3-EGFP)1Dean/J
71 strains use this protocol