Protocol 22204: QPCR Assay - Generic Neo Quantitative PCR-QPCR- 1.2
Version 8.0

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
13741 Fluorophore-1 CAT CGC ATC GAG CGA GCA CGT ACT Quencher-1 Tg Probe
oIMR1080 CGG CTG CAT ACG CTT GAT C Transgene Forward A
oIMR1081 CGA CAA GAC CGG CTT CCA T Transgene Reverse A
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
oIMR1080 0.40 uM
oIMR1081 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
004166 129-Itgb5tm1Des/J
008668 129S-Hba-a1tm1Led/J
005032 B6 x B6Ei.129(Cg)-Bmp8btm1Blh/EiJ
028635 B6.129(Cg)-Cntnap2tm2Pele/J
029902 B6.129-Ptprctm1Mak/Mmjax
034226 B6.129-Rab25tm1Jrgo/J
003124 B6.129-Ucp1tm1Kz/J
010725 B6.129-Ugt1tm1Rhtu/J
017976 B6.129-Ulk1tm1Thsn/J
029253 B6.129P2(Cg)-Card6tm1.1Aldu/MakJ
024630 B6.129P2-Lcn2tm1Aade/AkiJ
024839 B6.129P2-Pzptm1Vln Mug1tm2Vln/J
023496 B6.129P3-Rasgrp3tm1Jstn/TbwnJ
034595 B6.129S-Capn1tm1Ahc/J
018311 B6.129S4-Catsper1tm1Clph/J
009340 B6.129S6(Cg)-Eif2ak3tm1Dron/HotaJ
017975 B6.129S6-Dag1tm5Kcam/J
011131 B6.129X1(Cg)-Arrb1tm1Jse/J
004225 B6.129X1-Zap70tm1Weis/J
033705 B6.Cg-Tg(Lck-RHO/ADRA1,-lacZ)B22-1Mik/J
033109 B6;129-Cdh2tm1(cre/ERT)Linli/J
024649 B6;129-Gdf7tm1Kng/J
035444 B6;129-Hspa5tm1Alee/J
006378 B6;129-Nrxn2tm3Sud/J
018437 B6;129-Pax2tm1Gdr/J
003284 B6;129S1-Il1raptm1Roml/J
019578 B6;129S1-Rnlstm1Gvd/J
002914 B6;129S4-Hexbtm1Rlp/J
023343 B6;129S4-Pcmt1tm1Scl/J
370476 B6J.129S1-Htttm1Mfc/190JChdi
034731 B6N(129S4)-Srd5a3tm1c(EUCOMM)Wtsi/J
034643 B6N.129-Irgm1tm1Gat/J
025330 B6N.129P2(Cg)-Igs13tm1Dolm Igs14tm1Dolm/J
032924 B6N.129X1(B6J)-Msratm1Jmo/RlevMmjax
024744 B6N.Cg-Idstm1Muen/J
008340 BKS.Cg-Leprdb Nos3tm1Unc/RhrsJ
002724 C.129S2(B6)-Cxcr2tm1Mwm/J
014593 C;129S4-Rag2tm1.1Flv Il2rgtm1.1Flv/J
017959 C57BL/6-Pmltm1(PML/RARA)Ley/J
007078 CByJ.129S2(B6)-Il6tm1Kopf/J
022760 MRL.Cg-Nos3tm1Unc Faslpr/J
006023 NOD.Cg-H2-Ab1tm1Gru Tg(CD4,HLA-DQA1,HLA-DQB1)N8Ell/EllJ
035185 STOCK Adgrl1tm2c(EUCOMM)Hmgu/SudJ
003810 STOCK Adrb1tm1Bkk Adrb2tm1Bkk/J
030647 STOCK Casrtm1Mrpk/J
024390 STOCK Hat1tm1Mrpa/J
003259 STOCK Igf1tm2Ts/ImJ
030855 STOCK Notch1tm2Rko Notch2tm3Grid Gt(ROSA)26Sortm9(CAG-tdTomato)Hze Tg(KRT5-cre/ERT2)2Ipc/JeswJ
027645 STOCK Rhotm1Jlem Tg(Rho*)2PRieke/J
027646 STOCK Rhotm1Jlem Tg(Rho*)5PRieke/J
027647 STOCK Rhotm1Jlem Tg(Rho*)STM2Rieke/J
027648 STOCK Rhotm1Jlem Tg(Rho*)TTM1Rieke/J
027649 STOCK Rhotm1Jlem Tg(Rho*)TTM4Rieke/J
033159 STOCK Utf1tm1Jw/Mmjax
54 strains use this protocol