Stock Number: 006497 Strain Name: B6.129-Skiltm2Spw/J

Allele: Skiltm2Spw Protocol Name: Skiltm2Spw Method: Standard PCR Version: 1.1 Created: 30-June -2008 Updated: 24-September-2009

Expected Results: Mutant = 700 bp Heterozygote = 437 bp and 700 bp Wild type = 437 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 oIMR6416 - AAT GTG AGC GAG TAA CAA CCC G - Mutant oIMR6417 - CCA ACC AAA CTC AAC CAT TAC ACC - Common oIMR6418 - TTC ACA GGA GGA CTG CCA TCA TCC - Wild type

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 3.36 - 3.36 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 oIMR6416 0.60 1.00 uM 0.60 20 uM oIMR6417 0.60 1.00 uM 0.60 20 uM oIMR6418 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 60 1 min - 4 72 1 min repeat steps 2-4 for 35 cycles 5 72 2 min - 6 10 - hold

This genotyping protocol is used for the following strains:   Stock Number Strain Name 006497 B6.129-Skiltm2Spw/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.