Stock Number: 002495 Strain Name: B6;129S4-Col1a1tm1Jae/J

Allele: Col1a1tm1Jae Protocol Name: Cola1tm1Jae Method: Restriction Enzyme Digest Version: 1.1 Created: 15-February -2001 Updated: 13-October -2009

Notes: Use Perfect Match© (Stratagene) added before Taq to delete non-specific bands. Expected Results: Mutant = 412 bp and 225 bp Heterozygote = 667 bp, 412 bp and 225 bp Wild type = 667 bp

Gel Image

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Gel Information: Separated by gel electrophoresis on a 3.0% agarose gel.**

Protocol Primers

Primer 5' Label Sequence 5' --> 3' 3' Label Primer Type oIMR0810 - TGG TTC TGG AAT GAG GAT GG - Common oIMR0811 - TGC CTC TGC TTC CTT AGT GC - Common

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 2.80 - 2.80 10 X PCR Buffer II (ABI) 1.20 0.89 X 1.20 25 mM MgCl2 0.72 1.33 mM 0.72 2.5 mM dNTP 0.96 0.18 mM 0.96 20 uM oIMR0810 0.80 1.19 uM 0.80 20 uM oIMR0811 0.80 1.19 uM 0.80 5 mM DNA Loading Dye 1.66 0.62 mM 1.66 1 U/ul Perfect Match (Stratagene) 1.00 0.07 U/ul 1.00 5 U/ul Taq DNA Polymerase 0.06 0.02 U/ul 0.06 DNA 3.50 - 3.50

Cycling  Step # Temp °C Time Note 1 94 3 min - 2 94 30 sec - 3 65 1 min - 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) 10 X Buffer 2.00 - 2.00 ddH2O 11.08 - 11.08 10 mg/ml BSA 0.12 - 0.12 Sph I 0.80 - 0.80 This genotyping protocol is used for the following strains:   Stock Number Strain Name 002495 B6;129S4-Col1a1tm1Jae/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.