Stock Number: 009089 Strain Name: B6.129S1(Cg)-Ndntm2Stw/J

Allele: Ndntm2Stw Protocol Name: Ndn (tm2Stw/J) Sep PCR Method: Separated PCR Version: 1.0 Created: 18-March -2010 Updated: 19-March -2010

Expected Results: Mutant = 230 bp Heterozygote = 230 bp and 437 bp Wild type = 437 bp

Gel Image

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Protocol Primers

Primer 5' Label Sequence 5' --> 3' 3' Label Primer Type 8474 - CAC TTC CTC TGC TGG TCT CC - Common 8475 - TCC TTC ACC AAC ACG TAC CA - Wild type Reverse oIMR9012 - GTT TTC CCA GTC ACG ACG TT - Mutant Reverse

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 3.96 - 3.96 5 U/ul Taq DNA Polymerase 0.06 0.03 U/ul 0.06 2.5 mM dNTP 0.96 0.20 mM 0.96 5 mM DNA Loading Dye 1.66 0.69 mM 1.66 25 mM MgCl2 0.96 2.00 mM 0.96 DNA 2.00 - 2.00 20 uM 8475 0.60 1.00 uM 0.60 10 X AB PCR BufferII 1.20 1.00 X 1.20 20 uM 8474 0.60 1.00 uM 0.60

Cycling  Step # Temp °C Time Note 1 94 3 min - 2 94 30 sec - 3 62 30 sec - 4 72 30 sec - 5 - - Go to step 2-4, 35 times 6 72 2 min - 7 10 - hold

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 3.96 - 3.96 10 X AB PCR BufferII 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 8474 0.60 1.00 uM 0.60 20 uM oIMR9012 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 009089 B6.129S1(Cg)-Ndntm2Stw/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 62 30 sec - 4 72 30 sec - 5 - - Go to step 2-4, 35 times 6 72 2 min - 7 10 - hold