Stock Number: 002122 Strain Name: B6.129P2-Tcrbtm1Mom Tcrdtm1Mom/J

Allele: Tcrdtm1Mom Protocol Name: Generic Neo Melt Curve Analysis Method: Melt Curve Analysis Version: 1.1 Created: 17-September-2008 Updated: 05-June -2011

Notes: Melting curve analysis is performed using a Roche Light Cycler 480. Expected Results: Mutant Tm = 90°C +/- 1.0°C Wild type Tm = 82°C+/- 1.0°C

Gel Image

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Protocol Primers

Primer 5' Label Sequence 5' --> 3' 3' Label Primer Type oIMR6916 - CTT GGG TGG AGA GGC TAT TC - Mutant Forward oIMR6917 - AGG TGA GAT GAC AGG AGA TC - Mutant Reverse oIMR8744 - CAA ATG TTG CTT GTC TGG TG - Internal Positive Control Forward oIMR8745 - GTC AGT CGA GTG CAC AGT TT - Internal Positive Control Reverse

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 2.20 - 2.20 2 X SYBR Green Master Mix (Roche) 6.00 1.00 X 6.00 20 uM oIMR6916 0.60 1.00 uM 0.60 20 uM oIMR6917 0.60 1.00 uM 0.60 20 ul oIMR8744 0.30 0.50 ul 0.30 20 uM oIMR8745 0.30 0.50 uM 0.30 DNA 2.00 - 2.00

Cycling  Step # Temp °C Time Note 1 94 5 min - 2 94 20 sec - 3 64 30 sec* * -0.5 C per cycle 4 72 35 sec repeat steps 2-4 for 12 cycles 5 94 20 sec - 6 58 30 sec - 7 72 35 sec repeat steps 5-7 for 25 cycles 8 72 2 min - 9 10 - hold

This genotyping protocol is used for the following strains:   Stock Number Strain Name 002120 B6.129P2-Tcrdtm1Mom/J 002122 B6.129P2-Tcrbtm1Mom Tcrdtm1Mom/J 002204 B6.129P2-Hbbtm1Unc/J 002222 B6.129S7-Twist1tm1Bhr/J 002594 B6;129S6-Gbatm1Nsb/J 002794 C.129S4-Tfap2atm1Jae/J 002810 B6CBA-Tg(HDexon1)62Gpb/1J 003295 B6;129-Ednrbtm1Ywa/J 003312 B6.129S7-Ngftm1Gne/J 004185 B6.129S4-Soat2tm1Far/J 004861 B6EiC3Sn-Ts(16C-tel)1Cje/DnJ 005032 B6 x B6Ei.129(Cg)-Bmp8btm1Blh/EiJ 005814 B6.129P2-Grm1tm1Dgen/J 005819 B6.129-Insl5tm1Dgen/J 005865 STOCK Tg(YACW408A5)1952Ricc/J 006378 B6;129-Nrxn2tm3Sud/J 006833 B6.129-Sgcbtm1Kcam/2J 006904 B6;129-Msctm1Eno/J 006955 B6.129S4(FVB)-Insrtm1Khn/J 007005 129S-Scg5tm1Led/J 008312 CBy.129P2(B6)-Tcrdtm1Mom/SzJ 009600 B6;129-Six2tm3(EGFP/cre/ERT2)Amc/J 009662 B6.129X1-Ppargc1atm1Dpk/J 009685 B6N.129S1(Cg)-Tnkstm1.1Yjc/J 010673 B6;129-Runx1tm3.1Spe/J 010744 STOCK Kcna2tm1Tem/J 010948 B6;129-Gabra1tm4Geh/J 011081 B6;129-Osmrtm1.1Nat/J 011082 B6;129-Edn2tm1.1Nat/J 012560 129-Zic4tm2Kjmi/J 012623 B6;129S4-Carftm1.1Aew/J 012627 B6.Cg-Stard4tm1.1Bres/J 012653 B6;129-Pax7tm1.1Fan/J 012820 B6;129-Fzd1tm1.1Nat/J 013717 B6.Cg-Slc20a1tm1.2Cmg/J 014099 STOCK Tg(Pmch-cre)1Lowl/J 014593 C;129S4-Rag2tm1.1Flv Il2rgtm1.1Flv/J 014594 C;129S4-Rag2tm1.1Flv Thpotm1.1(TPO)Flv Il2rgtm1.1Flv/J 016264 129S6-Fmn2tm1Led/RwwJ 016835 B6;129S4-L3mbtl1tm1.1Haho/J 017615 C.129(B6)-Trp53bp2tm1Cdlo/J 017990 STOCK Fkbp4tm1Dvds/J 018163 B6N.129-Ephb6tm1Jwu/J 018164 B6.129S6-Pdcd4tm1Yhcn/J 018437 B6;129-Pax2tm1Gdr/J 018648 B6.129S6(Cg)-Itgb2tm1.1Mskim/J 018773 129-Sult2b1tm1Rus/J 020943 B6;129S4-Atmtm1Bal/J 021078 B6N.129S1-Mrgprb4tm4.1(flpo)And/J 021202 B6.Cg-Psme1/Psme2tm1Tchi/J 021451 B6.129S4-Catsperdtm1.1Clph/J 021485 B6N(Cg)-Cptm1b(KOMP)Wtsi/J 021752 B6N(Cg)-Tpgs2tm1.1(KOMP)Vlcg/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.