Stock Number: 002935 Strain Name: FVB.129S2(B6)-Ccnd1tm1Wbg/J

Allele: Ccnd1tm1Wbg Protocol Name: Ccndtm1Wbg Method: Separated PCR Version: 2.1 Created: 14-August -2008 Updated: 04-November -2009

Expected Results: Mutant = 391 bp Heterozygote = 224 bp and 391 bp Wild type = 224 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 oIMR0329 - TAG CAG AGA GCT ACA GAC TTC G - Common oIMR0330 - CTC CGT CTT GAG CAT GGC TC - Wild type oIMR0331 - CTA GTG AGA CGT GCT ACT TC - Mutant

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 4.20 - 4.20 10 X AB PCR BufferII 0.96 0.80 X 0.96 25 mM MgCl2 0.96 2.00 mM 0.96 2.5 mM dNTP 0.96 0.20 mM 0.96 20 uM oIMR0329 0.60 1.00 uM 0.60 20 uM oIMR0331 0.60 1.00 uM 0.60 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 30 sec - 3 64 45 sec - 4 72 1 min repeat steps 2-4 for 35 cycles 5 72 2 min - 6 10 - hold

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 4.20 - 4.20 10 X AB PCR BufferII 0.96 0.80 X 0.96 25 mM MgCl2 0.96 2.00 mM 0.96 2.5 mM dNTP 0.96 0.20 mM 0.96 20 uM oIMR0329 0.60 1.00 uM 0.60 20 uM oIMR0330 0.60 1.00 uM 0.60 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 This genotyping protocol is used for the following strains:   Stock Number Strain Name 002537 B6;129S2-Ccnd1tm1Wbg/J 002935 FVB.129S2(B6)-Ccnd1tm1Wbg/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.

Cycling  Step # Temp °C Time Note 1 94 3 min - 2 94 30 sec - 3 64 45 sec - 4 72 1 min repeat steps 2-4 for 35 cycles 5 72 2 min - 6 10 - hold