Protocol 22182: QPCR Assay - Generic GFP/EGFP qPCR
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
Kapa Probe Fast QPCR 1.00 X
ddH2O
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-Otx2tm2(cre/ERT2)Tlam/J
003960 129S6-Tg(Prnp-GFP/cre)1Blw/J
032051 B6(129S4)-Kittm1.1(cre)Jmol/J
033943 B6.129(C)-Bdnftm1.1Bail/KmrtJ
035371 B6.129(Cg)-Msi2tm1.1Geno/ReyaJ
004218 B6.129(ICR)-Tg(CAG-ECFP)CK6Nagy/J
030952 B6.129-Gt(ROSA)26Sortm5.1(CAG-Sun1/sfGFP)Nat/MmbeJ
032536 B6.129P2-Aviltm2(cre)Fawa/J
036013 B6.Cg-Axin2tm1.1Rva/J
033942 B6.Cg-Bdnftm2.1Kmrt/J
030305 B6.Cg-Eef1a1tm1Rck/J
028719 B6.Cg-Relbtm1Ukl/J
014135 B6.Cg-Tg(Fos/EGFP)1-3Brth/J
006864 B6.Cg-Tg(Ins1-EGFP)1Hara/J
031696 B6.Cg-Tg(Itgax-FCER1A,-EGFP)1Maur/J
007902 B6.Cg-Tg(RP23-268L19-EGFP)2Mik/J
018055 B6.FVB-Tg(H2-K-S100a9,GFP)1Gabr/J
021039 B6;129-Gt(ROSA)26Sortm5(CAG-Sun1/sfGFP)Nat/J
017495 B6;129S7-Crim1tm1(GFP/cre/ERT2)Pzg/J
030537 B6;129S7-Drd2tm1.1Jtw/J
033639 B6;CBA-Tg(Gnrh1-EGFP)51Sumo/J
033357 B6;CBA-Tg(Plp1-EGFP)10Wmac/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
028356 B6N.Cg-Tg(Sftpc,-EGFP)1Dobb/J
019108 C57BL/6-Efna3tm1Ebp/J
036197 C57BL/6J-Nr1i2em1Neg/J
007677 CB6-Tg(Gad1-EGFP)G42Zjh/J
007076 CByJ.B6-Tg(UBC-GFP)30Scha H2b/J
008450 FVB-Tg(CAG-luc,-GFP)L2G85Chco/J
003718 FVB-Tg(GadGFP)45704Swn/J
003516 FVB.Cg-Tg(CAG-EGFP)B5Nagy/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 Adgra3tm1.1(HBEGF,-cre/ERT2)Pac/Mmjax
032003 STOCK Grm7Tg(SMN2)89Ahmb Smn1tm1Msd Tg(Hlxb9-GFP)1Tmj Tg(SMN2*delta7)4299Ahmb/J
017530 STOCK Igs2tm2(ACTB-tdTomato,-EGFP)Luo Trp53tm1Tyj Nf1tm1Par/J
013751 STOCK Igs2tm2(ACTB-tdTomato,-EGFP)Luo/J
034694 STOCK Igs7tm86(tetO-CiVS/pHluorin)Hze/VpiJ
018322 STOCK Tg(Cp-EGFP)25Gaia/ReyaJ
023345 STOCK Tg(Pgk1-Ccnb1/EGFP)1Aklo/J
42 strains use this protocol