Stock Number: 002665 Strain Name: B6.129S2-Cd8atm1Mak/J

Allele: Cd8atm1Mak Protocol Name: Cd8atm1Mak Method: Standard PCR Version: 4.0 Created: 16-February -2012 Updated: 16-February -2012

Expected Results: Mutant = 343 bp Heterozygote = 265 bp and 343 bp Wild type = 265 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 oIMR1098 - GAC CTG GTA TGT GAA GTG TTG G - Common oIMR1099 - ACA TCA CCG AGT TGC TGA TG - Wild type oIMR6828 - CAT AGC GTT GGC TAC CCG TG - Mutant

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 3.00 - 3.00 5 X KAPA LR HotStart Buffer 2.40 1.00 X 2.40 25 mM MgCl2 0.96 2.00 mM 0.96 2.5 mM dNTP 0.96 0.20 mM 0.96 20 uM oIMR1098 0.30 0.50 uM 0.30 20 uM oIMR1099 0.30 0.50 uM 0.30 20 uM oIMR6828 0.30 0.50 uM 0.30 5 mM DNA Loading Dye 1.66 0.69 mM 1.66 2.5 U/ul KAPA LR HotStart Taq 0.12 0.03 U/ul 0.12 DNA 2.00 - 2.00

Cycling  Step # Temp °C Time Note 1 94 2 min - 2 94 20sec - 3 65 15sec -1.5 C per cycle 4 68 10sec - 5 - - repeat step 2-4 for 10 cycles 6 94 15sec - 7 50 15sec - 8 72 10sec - 9 - - repeat step 6-8 for 28 cycles 10 72 2 min - 11 10 - hold

This genotyping protocol is used for the following strains:   Stock Number Strain Name 002664 B6;129S-Cd4tm1Mak Cd8atm1Mak/J 002665 B6.129S2-Cd8atm1Mak/J 005513 NOD.129S2(B6)-Cd8atm1Mak/DvsJ 007071 CByJ.129S2(B6)-Cd8atm1Mak/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.