Stock No: 013087
Protocol 29086: Standard PCR Assay - Generic Neo
Version 2.3

Notes

Melting curve analysis is performed using a Roche Light Cycler 480.

PLEASE NOTE: This assay will distinguish heterozygous from homozygous Tcrd<tm1Mom> mutant mice.

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

Mutant Tm = 90°C +/- 1.0°C
Wild type Tm = 82°C+/- 1.0°C

Mut = 280 bp

IPC = 206 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR6916 CTT GGG TGG AGA GGC TAT TC Mutant Forward A Neo
oIMR6917 AGG TGA GAT GAC AGG AGA TC Mutant Reverse A Neo
oIMR8744 CAA ATG TTG CTT GTC TGG TG Internal Positive Control Forward A
oIMR8745 GTC AGT CGA GTG CAC AGT TT 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
oIMR6916 0.50 uM
oIMR6917 0.50 uM
oIMR8744 0.50 uM
oIMR8745 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
025451 129-Iqgap1tm1Aber/VsJ
017631 129-Nrarptm1Grid/J
012560 129-Zic4tm2Kjmi/J
024906 129-Dp(16Tiam1-Il10rb)8Yey/J
028028 129S(B6N)-Cdk1tm1Eddy/J
024634 129S(Cg)-Tgfbr2tm1.1Hcd/J
024525 129S-Gnai3tm1Lbi Gnai1tm1Lbi/Mmjax
007005 129S-Scg5tm1Led/J
018424 129S-Slc26a4tm1Egr/AjgJ
016264 129S6-Fmn2tm1Led/RwwJ
005032 B6 x B6Ei.129(Cg)-Bmp8btm1Blh/EiJ
029290 B6(Cg)-Sgtatm1.1Drmcr/J
021072 B6(Cg)-Slc6a8tm1.2Clar/J
024469 B6(SJL)-Lrrk2tm3.1Mjfa/J
021828 B6(SJL)-Lrrk2tm3.1Mjff/J
025930 B6(SJL)-Pik3cbtm1.1Jdo/J
023313 B6.129(Cg)-Homer2tm1Mhd/PfwJ
008320 B6.129(Cg)-Leprtm2(cre)Rck/J
029799 B6.129-Ank3tm1Bnt/J
023853 B6.129-Chgatm1Gnh/J
004276 B6.129-Figntm1Frk/Frk
005819 B6.129-Insl5tm1Dgen/J
028890 B6.129-Ncor1tm1Rsd/J
022996 B6.129-Nox4tm1Kkr/J
025633 B6.129-Pdk4tm1Rhar/J
028765 B6.129-Pfdn1Gt(GTR1.3)1Rul/WkhnJ
002725 B6.129-Ptprctm1Holm/J
034226 B6.129-Rab25tm1Jrgo/J
006833 B6.129-Sgcbtm1Kcam/2J
023309 B6.129-Stat2tm1Shnd/J
022364 B6.129P2(SJL)-Ntrk3tm1Ddg/J
022499 B6.129P2-Adora2btm1Till/J
023726 B6.129P2-C1qatm1.1Tenn/J
029905 B6.129P2-Ctla4tm1Mak/Mmjax
005814 B6.129P2-Grm1tm1Dgen/J
002204 B6.129P2-Hbbtm1Unc/J
029904 B6.129P2-Msh2tm1Mak/Mmjax
002122 B6.129P2-Tcrbtm1Mom Tcrdtm1Mom/J
002120 B6.129P2-Tcrdtm1Mom/J
005845 B6.129P2-Thbs4tm1Dgen/J
034595 B6.129S-Capn1tm1Ahc/J
027187 B6.129S-Lnpeptm1Ske/J
023209 B6.129S-Sfrp1tm1Jrbn/PcmaJ
030536 B6.129S1-Tfe3tm1Est/Mmjax
021055 B6.129S2(Cg)-Bdnftm1Krj/J
005027 B6.129S2-Il10rbtm1Agt/J
030087 B6.129S4(C)-Miftm1Dvd/MgadJ
006955 B6.129S4(FVB)-Insrtm1Khn/J
004163 B6.129S4-Cdk5r1tm1Lht/J
002781 B6.129S4-Cdkn1btm1Mlf/J
004185 B6.129S4-Soat2tm1Far/J
018648 B6.129S6(Cg)-Itgb2tm1.1Mskim/J
017975 B6.129S6-Dag1tm5Kcam/J
018164 B6.129S6-Pdcd4tm1Yhcn/J
025929 B6.129S7(Cg)-Pik3catm1Jdo/J
008683 B6.129S7-Cacna1atm3Hzo/J
003312 B6.129S7-Ngftm1Gne/J
022767 B6.129S7-Slc7a2tm1Clm/LellJ
002222 B6.129S7-Twist1tm1Bhr/J
028953 B6.129X1-Id4tm1Mais/Mmjax
034240 B6.Cg-Gt(ROSA)26Sortm1(CAG-Eos*/Calm*)Mrc/J
030743 B6.Cg-Gt(ROSA)26Sortm1(CAG-Pycard/mCitrine*,-CD2*)Dtg/J
006513 B6.Cg-Ighmbp2nmd-2J Tg(Ttn-Ighmbp2)45Cx/Cx
004989 B6.Cg-Mbd4tm1Wed/J
027748 B6.Cg-Mybl1tm1Epr/J
029031 B6.Cg-Pabpc1ltm1.1Esel/J
013717 B6.Cg-Slc20a1tm1.2Cmg/J
012627 B6.Cg-Stard4tm1.1Bres/J
033705 B6.Cg-Tg(Lck-RHO/ADRA1,-lacZ)B22-1Mik/J
008135 B6.Cg-Tg(THY1-SNCA*A53T)M53Sud/J
033109 B6;129-Cdh2tm1(cre/ERT)Linli/J
011082 B6;129-Edn2tm1.1Nat/J
003295 B6;129-Ednrbtm1Ywa/J
027583 B6;129-Esyt1tm1Sud Esyt2tm1Sud/J
012820 B6;129-Fzd1tm1.1Nat/J
010948 B6;129-Gabra1tm4Geh/J
012894 B6;129-Macf1tm1Efu/J
006904 B6;129-Msctm1Eno/J
006378 B6;129-Nrxn2tm3Sud/J
011081 B6;129-Osmrtm1.1Nat/J
012653 B6;129-Pax7tm1.1Fan/J
025740 B6;129-Prg4tm2Mawa/J
010673 B6;129-Runx1tm3.1Spe/J
009600 B6;129-Six2tm3(EGFP/cre/ERT2)Amc/J
018773 B6;129-Sult2b1tm1Rus/J
002121 B6;129P-Tcrbtm1Mom Tcrdtm1Mom/J
006386 B6;129P2-Syt1tm5Sud/J
026012 B6;129P2-Uts2rtm1Djbe/J
024003 B6;129S-Matn1tm1Goe/J
027581 B6;129S-Nfatc4tm1Grc/J
003284 B6;129S1-Il1raptm1Roml/J
029304 B6;129S2-Syngap1tm2Geno/RumbJ
012623 B6;129S4-Carftm1.1Aew/J
016835 B6;129S4-L3mbtl1tm1.1Haho/J
003749 B6;129S5-Pak2Gt(OST104058)Lex/J
002594 B6;129S6-Gbatm1Nsb/J
027102 B6;129X1-Lhcgrtm1Zmlei/J
026526 B6;SJL-Akap6tm1.1Mskf/Mmjax
007266 B6Ei.129S1(Cg)-Zfpm2tm2Sho/EiJ
027410 B6J.129S1-Htttm1Mfc/190ChdiJ
370476 B6J.129S1-Htttm1Mfc/190JChdi
030975 B6J.129S7-Pamtm1Jep/Mmjax
034731 B6N(129S4)-Srd5a3tm1c(EUCOMM)Wtsi/J
034643 B6N.129-Irgm1tm1Gat/J
021078 B6N.129S1-Mrgprb4tm4.1(flpo)And/J
029976 B6N.129S6-Retnlatm1Tliu/J
032924 B6N.129X1(B6J)-Msratm1Jmo/RlevMmjax
007552 B6SJL-Chrdtm1Emdr/J
021568 BXSB.129(Cg)-Il21rtm1Wjl/DcrJ
017615 C.129(B6)-Trp53bp2tm1Cdlo/J
005700 C.129P2-Cxcr6tm1Litt/J
002794 C.129S4-Tfap2atm1Jae/J
014593 C;129S4-Rag2tm1.1Flv Il2rgtm1.1Flv/J
023520 C57BL/6-Bcrtm1(BCR/ABL)Tsr/J
029836 C57BL/6-Gt(ROSA)26Sortm1(CAG-SPAST*C448Y)Baas/J
009645 CB6-Isl1tm1Tmj/J
008312 CBy.129P2(B6)-Tcrdtm1Mom/SzJ
003219 D2.129P2(B6)-Nr5a1tm1Klp/EiJ
012440 DBA/2J-Tg(RP24-180N9)2Kjn/Kjn
026396 FVB.129S2(B6)-Bcrtm1Hkp/J
022036 FVB.129X1(Cg)-Atp2a2tm1Ges/Mmjax
027670 FVB/NJ-Tg(Snca)2Nbm/J
022395 NOD.129P2(B6)-Tcrdtm1Mom/JsdJ
004352 NOD.129S1(B6)-Ifngr2tm1Pbro/DvsJ
035185 STOCK Adgrl1tm2c(EUCOMM)Hmgu/SudJ
026229 STOCK Akap12tm1Ihg Rb1tm2Brn Tg(Pbsn-cre)4Prb/J
025665 STOCK Asxl1tm1.1Iaai/J
026629 STOCK Cyp26b1tm1Dcp/J
026199 STOCK F8tm1.1Rmnt/J
017990 STOCK Fkbp4tm1Dvds/J
027233 STOCK Flnbtm1Vshn/J
028096 STOCK Gata6tm1Hadj/J
029812 STOCK Gt(ROSA)26Sortm1(RARA*)Soc/HsvJ
023314 STOCK Homer3tm1Mhd/PfwJ
022976 STOCK Igs2tm1(ACTB-EGFP,-tdTomato)Zng/J
003534 STOCK Inpp5dtm1Dmt/J
029787 STOCK Klf5tm1Jaw/J
023916 STOCK Lin28atm1.1Gqda/J
017508 STOCK Mir137tm1Mtm/Mmjax
013087 STOCK Mir7-2tm1Mtm/Mmjax
030357 STOCK Pou4f2tm1(cre)Bnt/J
022972 STOCK Pros1tm1Grl/J
025693 STOCK Tg(CAG-GFP,-mCherry/Pxn)#Mgin/KleyJ
007731 STOCK Tg(Col2a1-Npr2*B1)2Gar/J
005865 STOCK Tg(YACW408A5)1952Ricc/J
145 strains use this protocol