Stock Number: 002082 Strain Name: 129S-Rb1tm1Tyj/J

Allele: Rb1tm1Tyj Protocol Name: NEOTD (Generic Neo) Method: Standard PCR Version: 1.1 Created: 28-May -2004 Updated: 29-October -2009

Expected Results: NEO (mutant) band = 280 bp Tcrd internal control = ~ 200 bp

Gel Image

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Gel Information: Separated by gel electrophoresis on a 1.5% agarose gel.

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 3.66 - 3.66 10 X PCR Buffer II (ABI) 1.20 1.00 X 1.20 25 mM MgCl2 0.96 2.00 mM 0.96 2.5 mM dNTP 0.96 0.20 mM 0.96 20 uM oIMR6916 0.45 0.75 uM 0.45 20 uM oIMR6917 0.45 0.75 uM 0.45 20 oIMR8744 0.30 0.50 0.30 20 uM oIMR8745 0.30 0.50 uM 0.30 5 mM DNA Loading Dye 1.66 0.69 mM 1.66 5 U/ul Taq DNA Polymerase 0.06 0.03 U/ul 0.06 DNA 2.00 - 2.00

Cycling  Step # Temp °C Time Note 1 94 3 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 002082 129S-Rb1tm1Tyj/J 002098 STOCK Tgfb1tm1Doe/J 002099 B6.129X1-Fostm1Pa/J 002102 B6.129S2-Rb1tm1Tyj/J 002119 B6;129P2-Tcrdtm1Mom/J 002120 B6.129P2-Tcrdtm1Mom/J 002121 B6;129P-Tcrbtm1Mom Tcrdtm1Mom/J 002122 B6.129P2-Tcrbtm1Mom Tcrdtm1Mom/J 002123 B6;129S-Dnmt1tm1Jae/J 002124 C;129S-Ngfrtm1Jae/J 002189 B6;129S7-Ass1tm1Bay/J 002198 B6.129S4-Dnmt1tm1Jae/J 002219 B6.129P2-Tgfatm1Ard/J 002222 B6.129S7-Twist1tm1Bhr/J 002265 B6;129S2-Bcl2tm1Sjk/J 002275 B6.129S4-Ntf3tm1Jae/J 002276 STOCK Ntf3tm1Jae/J 002293 B6;129X1-Fostm1Pa/J 002362 B6.129P2-Camk2atm1Sva/J 002426 B6;129S4-Insrtm1Dac/J 002444 B6;129S-Syn1tm1Sud/J 002478 B6;129S-Syt1tm1Sud/J 002481 B6;129S2-Ntrk3tm1Bbd/J 002516 129-Edn3tm1Ywa/J 002544 B6;129S2-Ntrk2tm1Bbd/J 002616 B6.129S7-Hbatm1Paz/J 002619 B6.129-Tgfb3tm1Doe/J 002719 B6.129S4-Wt1tm1Jae/J 002850 B6;129S4-Nfkbiatm1Bal/J 002851 B6;129S4-Relatm1Bal/J 002900 FVB.129S2(B6)-Rb1tm1Tyj/J 002908 129-Col4a3tm1Dec/J 002991 B6.129-Ascl1tm1And/J 003098 B6.129S2-Ntrk2tm1Bbd/J 003123 129S;ICR-Vhltm1Bjg/J 003178 B6.129-Arnttm1Mcs/J 003201 129/Sv-Csktm1Sor/J 003259 STOCK Igf1tm2Ts/ImJ 003316 B6.129P2-Gcktm1Efr/J 003343 B6.129S2-En1tm1Alj/J 003447 CBy.129P2(B6)-Tcrbtm1Mom/SzJ 003448 CByJ.129P2(B6)-Tcrdtm1Mom/Sz 003463 STOCK Agatm1Vk/J 003568 B6.129S7-Trp63tm2Brd/J 003576 STOCK Grm4tm1Hpn/J 003652 C57BL/6-Ptprcaptm1Tho/J 003694 B6;129S-Vamp8Gt(OST20346)Lex/J 003696 B6;129S-Thbs3Gt(OST10612)Lex/J 003725 B6.129-Gabrdtm1Geh/J 003727 B6.129P2-Cd38tm1Lnd/J 003781 B6.129P2-Clns1atm1Kwn/J 004038 B6.129S4-Zp3tm1Dean/J 004067 B6.129S4-Ep300tm1Dli/J 004185 B6.129S4-Soat2tm1Far/J 004234 B6;129S-Fgfr3tm1Dor/J 004267 B6.129S6-Dnmt3ltm1Bes/J 004373 B6;129S6-Apaf1tm1Her/J 004374 129-Npr1tm1Gar/J 004514 129S-Tnp2tm1Mzh/J 004861 B6EiC3Sn-Ts(16C-tel)1Cje 004943 NOD.Cg-Selltm1Tft Itgb7tm1Cgn/LtJ 005814 B6.129P2-Grm1tm1Dgen/J 005819 B6.129-Insl5tm1Dgen/J 005850 B6.129P2-Mapkapk2tm1Dgen/J 005865 STOCK Tg(YACW408A5)1952Ricc/J 006330 NOD.129S2(B6)-Cd1d1/Cd1d2tm1Spb/SbwJ 006833 B6.129-Sgcbtm1Kcam/2J 006838 D2.129S1(Cg)-Zfpm2tm1Sho/EiJ 006904 B6;129-Msctm1Eno/J 006908 B6.Cg-Ikbketm1Tman/J 006944 B6.129-Mgl1tm1Hed/J 007005 129S-Scg5tm1Led/J 007065 B6;129S4-Glra2tm1Clc/J 007266 B6Ei.129S1(Cg)-Zfpm2tm2Sho/EiJ 007664 129S-Efnb1tm1Sor/J 008078 B6;129S4-Tcf3tm5Zhu/J 008120 B6.129S4-Timp2tm1Pds/J 008138 STOCK Stx1btm1Sud/J 008275 B6.129S6-Sgcatm2Kcam/J 008385 B6.129-Leprtm2Mgmj/J 008419 B6;129S4-Pkd1l3tm1Sul/J 008445 B6.129S-Fn1tm1Hyn/2J 008518 B6.129-Leprtm1Mgmj/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.