Stock Number: 006494 Strain Name: B6CBA-Tg(HDexon1)62Gpb/3J

Allele: Pde6brd1 Protocol Name: Generic Pde6b Method: Standard PCR Version: 1.1 Created: 15-October -2007 Updated: 24-September-2009

Notes: 10X AM Buffer: 67mM Tris.HCL pH8.8, 16.6mM NH4SO4, 2.0mM MgCl2, 0.17mg/ml BSA. Dissolve in 9 ml TE, pH to 8.8, then bring final volume to 10 ml with TE (NOT water!) Filter sterilize. Add 2-mercaptoethanol to 10 mM prior to use. Size product on ABI3730. Expected Results: Mutant = 560 bp Heterozygote = 240 bp and 560 bp Wild type= 240 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 oIMR2093 - AAG CTA GCT GCA GTA ACG CCA TTT - Mutant oIMR2094 - ACC TGC ATG TGA ACC CAG TAT TCT ATC - Wild type oIMR2095 - CTA CAG CCC CTC TCC AAG GTT TAT AG - Common

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 3.90 - 3.90 10 X PCR Buffer II (ABI) 1.20 1.00 X 1.20 25 mM MgCl2 0.72 1.50 mM 0.72 2.5 mM dNTP 0.96 0.20 mM 0.96 20 uM oIMR2093 0.48 0.80 uM 0.48 20 uM oIMR2094 0.48 0.80 uM 0.48 20 uM oIMR2095 0.48 0.80 uM 0.48 5 mM DNA Loading Dye 1.66 0.69 mM 1.66 5 U/ul Taq DNA Polymerase 0.12 0.05 U/ul 0.12 DNA 2.00 - 2.00

Cycling  Step # Temp °C Time Note 1 94 5 min - 2 94 30 sec - 3 65 30 sec - 4 72 1.5 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 001924 B6EiC3Sn a/A-Ts(1716)65Dn 002810 B6CBA-Tg(HDexon1)62Gpb/1J 004808 STOCK Mapttm1(EGFP)Klt Tg(MAPT)8cPdav/J 004828 FVB.129P2-Pde6b+ Tyrc-ch/AntJ 004850 B6EiC3Sn-Rb(12.Ts171665Dn)2Cje/CjeDn 004861 B6EiC3Sn-Ts(16C-tel)1Cje 005965 STOCK Tg(Pomc1-cre)16Lowl/J 006494 B6CBA-Tg(HDexon1)62Gpb/3J 006554 B6SJL-Tg(APPSwFlLon,PSEN1*M146L*L286V)6799Vas/J 006576 B6.FVB-Tg(GNAT2-Dta)98Wwk/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.