Stock Number:002900
Strain Name:FVB.129S2(B6)-Rb1tm1Tyj/J
Rb1tm1Tyj
NEOTD (Generic Neo)
Standard PCR
4.0
21-September-2011 21-September-2011
Notes:

This assay does not work well without the use of a Hotstart Taq polymerase.

For any mutation other than Tcrdtm1Mom mutants, this assay will not distinguish between heterozygotes and homozygotes.
Expected Results: NEO (mutant) band = 280 bp
Tcrd internal positive control = ~ 200 bp
Gel Image 
Image Image file
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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
Reaction/Components 
Reaction Component Volume (µl) Final
Concentration		 Total Volume (µl)
10 X KAPA Rxn Buffer 1.20 1.00 X 1.20
20 uM oIMR6916 0.60 1.00 uM 0.60
20 uM oIMR8745 0.30 0.50 uM 0.30
20 uM oIMR6917 0.60 1.00 uM 0.60
ddH2O 4.32 - 4.32
2.5 mM dNTP 0.96 0.20 mM 0.96
5 mM DNA Loading Dye 1.66 0.69 mM 1.66
DNA 2.00 - 2.00
20 uM oIMR8744 0.30 0.50 uM 0.30
5 U/ul KAPATaq DNA Polymerase 0.06 0.03 U/ul 0.06
Cycling 
Step # Temp °C Time Note
1 94 3 min -
2 94 20sec -
3 64 30sec -0.5 C per cycle
4 72 30sec repeat steps 2-4 for 12 cycles
5 94 20sec -
6 58 30sec -
7 72 35sec repeat steps 5-7 for 25 cycles
8 72 2min -
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
002121 B6;129P-Tcrbtm1Mom Tcrdtm1Mom/J
002123 B6;129S-Dnmt1tm1Jae/J
002124 C;129S-Ngfrtm1Jae/J
002189 B6;129S7-Ass1tm1Bay/J
002198 B6.129S4-Dnmt1tm1Jae/J
002204 B6.129P2-Hbbtm1Unc/J
002219 B6.129P2-Tgfatm1Ard/J
002221 B6;129S-Twist1tm1Bhr/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
002771 B6.129S2-Tlx1tm1Sjk/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 129S-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
004861 B6EiC3Sn-Ts(16C-tel)1Cje/DnJ
006330 NOD.129S6(B6)-Cd1d1/Cd1d2tm1Spb/SbwJ
006908 B6.Cg-Ikbketm1Tman/J
006944 B6.129-Clec10atm1Hed/J
008078 B6;129S4-Tcf3tm5Zhu/J
008312 CBy.129P2(B6)-Tcrdtm1Mom/SzJ
009340 STOCK Eif2ak3tm1Dron/HotaJ
017832 B6.129-Rac3tm1Hkp/J
018154 B6N.129S2-Fyntm1Rmp/J
Genotyping resources and troubleshooting
This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.