Stock Number: 006775 Strain Name: NOD.Cg-Foxp3sf/DoiJ

Allele: Foxp3sf Protocol Name: Foxp3sf Method: Separated PCR Version: 1.1 Created: 04-December -2003 Updated: 14-September-2010

Notes: This assay does not work well without the use of a Hotstart (We are using Taq Start Antibody mixed 1:1 with Taq polymerase). Use Perfect Match© (Stratagene) added before Taq to delete non-specific bands. Be aware that this genotyping assay has not always worked consistently well for The Jackson Laboratory's genotyping laboratory. Expected Results: Mutant = ~708 bp Wildtype = ~708 bp

Gel Image

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Gel Information: Separated by gel electrophoresis on a 1.5% agarose gel. Do not pool as both bands are approximately

Protocol Primers

Primer 5' Label Sequence 5' --> 3' 3' Label Primer Type oIMR1571 - CTC AGG CCT CAA TGG ACA AG - Wild type Forward oIMR1572 - TCA GGC CTC AAT GGA CAA AA - Mutant Forward oIMR1573 - CAT CGG ATA AGG GTG GCA TA - Common

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 3.58 - 3.58 10 X AB PCR BufferII 1.20 1.00 X 1.20 25 mM MgCl2 1.20 2.50 mM 1.20 2.5 mM dNTP 0.96 0.20 mM 0.96 20 uM oIMR1572 0.60 1.00 uM 0.60 20 uM oIMR1573 0.60 1.00 uM 0.60 5 mM DNA Loading Dye 1.66 0.69 mM 1.66 1 U/ul Perfect Match (Stratagene) 0.10 0.01 U/ul 0.10 5 U/ul Taq DNA Polymerase + TaqStart AB (1:1) 0.10 0.01 U/ul 0.10 DNA 2.00 - 2.00

Cycling  Step # Temp °C Time Note 1 94 3 min - 2 94 30 sec - 3 67 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) ddH2O 3.58 - 3.58 10 X AB PCR BufferII 1.20 1.00 X 1.20 25 mM MgCl2 1.20 2.50 mM 1.20 2.5 mM dNTP 0.96 0.20 mM 0.96 20 uM oIMR1571 0.60 1.00 uM 0.60 20 uM oIMR1573 0.60 1.00 uM 0.60 5 mM DNA Loading Dye 1.66 0.69 mM 1.66 1 U/ul Perfect Match (Stratagene) 0.10 0.01 U/ul 0.10 5 U/ul Taq DNA Polymerase + TaqStart AB (1:1) 0.10 0.01 U/ul 0.10 DNA 2.00 - 2.00 This genotyping protocol is used for the following strains:   Stock Number Strain Name 004088 B6.Cg-Foxp3sf/J 006775 NOD.Cg-Foxp3sf/DoiJ 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 68.9 1 min - 4 72 1 min repeat steps 2-4 for 35 cycles 5 72 2 min - 6 10 - hold