Protocol 21500: QPCR Assay - Fluorescent Proteins -- Generic GFP
Version 15.0

Notes

Taqman qPCR protocols are run on an ABI 7500, 7700, 7900 or the Roche LightCycler
480. 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
10201 AGT GCT TCA GCC GCT ACC Transgene Forward A GFP
10202 GAA GAT GGT GCG CTC CTG Transgene Reverse A GFP
13731 Fluorophore-1 TTC AAG TCC GCC ATG CCC GAA Quencher-1 Tg Probe GFP
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
10201 0.40 uM
10202 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
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
032051 B6(129S4)-Kittm1.1(cre)Jmol/J
035371 B6.129(Cg)-Msi2tm1.1Geno/ReyaJ
004218 B6.129(ICR)-Tg(CAG-ECFP)CK6Nagy/J
030305 B6.Cg-Eef1a1tm1Rck/J
022486 B6.Cg-Ptprca Tg(UBC-PA-GFP)1Mnz/J
014135 B6.Cg-Tg(Fos/EGFP)1-3Brth/J
006864 B6.Cg-Tg(Ins1-EGFP)1Hara/J
005630 B6.Cg-Tg(Thy1-EYFP)15Jrs/J
005738 B6.FVB-Tg(tetO-EGFP,-Tgfbr2)8Mcle/J
017495 B6;129S7-Crim1tm1(GFP/cre/ERT2)Pzg/J
030537 B6;129S7-Drd2tm1.1Jtw/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
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
012350 B6;SJL-Tg(Thy1-COP4*H134R/EYFP)20Gfng/J
023520 C57BL/6-Bcrtm1(BCR/ABL)Tsr/J
006567 C57BL/6-Tg(CAG-EGFP)131Osb/LeySopJ
005070 C57BL/6-Tg(Csf1r-EGFP-NGFR/FKBP1A/TNFRSF6)2Bck/J
004353 C57BL/6-Tg(UBC-GFP)30Scha/J
007076 CByJ.B6-Tg(UBC-GFP)30Scha H2b/J
005515 FVB-Tg(ITGAM-DTR/EGFP)34Lan/J
008173 NOD.Cg-Tg(Ins1-EGFP)1Hara/QtngJ
009422 NOD.Cg-Tg(Itgax-Venus)1Mnz/QtngJ
008547 NOD.FVB-Tg(ITGAM-DTR/EGFP)34Lan/JdkJ
032003 STOCK Grm7Tg(SMN2)89Ahmb Smn1tm1Msd Tg(Hlxb9-GFP)1Tmj Tg(SMN2*delta7)4299Ahmb/J
034694 STOCK Igs7tm86(tetO-CiVS/pHluorin)Hze/VpiJ
025855 STOCK Ptprca Lag3tm1Doi Tg(CAG-luc,-GFP)L2G85Chco Thy1a/J
005854 STOCK Tg(Cp-EGFP)25Gaia/J
018322 STOCK Tg(Cp-EGFP)25Gaia/ReyaJ
38 strains use this protocol