Stock Number: 000643 Strain Name: DW/J Mlphln Pou1f1dw/J

Allele: Pou1f1dw Protocol Name: Pou1f1dw Method: End Point Analysis Version: 1.1 Created: 19-February -2007 Updated: 24-September-2009

Notes: End point analysis is done using Amplifluor SNPS Genotyping System from Millipore. End point analysis protocols are run on an ABI 7000, 7500, or the Roche LightCycler 480

Sequence: TTGTTCATAGACAAAGATTTGGCTTTCACGTGTGCTTTTAGAATCTATGTTACCTCTTCA CGGTAATATTTAGAAAGTTAAGTTTCATCTTATTCTTCTAAGAACTTCATTGAAAGAAA CAAAATTTTATCCCAACTTCTCCCCTCACACTCCACCCCTTCCTCCCCAGTGTAGCTGC TAAGGATGCTCTGGAGAGACACTTCGGGGAGCACAGCAAGCCTTCCTCGCAGGAGAT CATGCGGATGGCTGAAGAACTGAATCTCGAGAAAGAAGTAGTACGAGTGTG(g/t)TTCTG CAACCGAAGGCAGAGAGAAAAACGAGTGAAAACAAGTCTCAATCAGAGCCTGTTCTC TATTTCCAAGGAACATCTAGAGTGCAGATAAGGCTGGCGTGTGATAGCCATGTGTGGTA GCTACTCCACTTCTTTCAGTTTTCAAGAAAAATCAGAAGTTAGTCACTTGGTTAAAAAC TAGATTGAATCAGTAACTCCTGCAGTTTTTG

Protocol Primers

Primer 5' Label Sequence 5' --> 3' 3' Label Primer Type oIMR4033 - GAA GGT CGG AGT CAA CGG ATT GAG AAA GAA GTA GTA CG - Mutant oIMR4034 - GAA GGT GAC CAA GTT CAT GCT GAG AAA GAA GTA GTA CG - Common oIMR4035 - CAG GCT CTG ATT GAG ACT TGT T - Wild type

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 4.60 - 4.60 20 X Ampliflour SNP FAM 0.50 1.00 X 0.50 20 X SNP JOE 0.50 1.00 X 0.50 20 X Primer Mix 0.50 1.00 X 0.50 10 X Rxn Mix S+ 1.00 1.00 X 1.00 2.5 mM dNTP 0.80 0.20 mM 0.80 5 U/ul Titanium Taq Pol. 0.10 0.05 U/ul 0.10 DNA 2.00 - 2.00

Cycling  Step # Temp °C Time Note 1 95 5 min - 2 94 30 sec - 3 55 30 sec - 4 72 40 sec repeat steps 2-4 for 40 cycles 5 72 2 min - 6 10 - hold

This genotyping protocol is used for the following strains:   Stock Number Strain Name 000643 DW/J Mlphln Pou1f1dw/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.