Stock Number: 004133 Strain Name: B6.129S7-Apptm1Dbo/J

Allele: Apptm1Dbo Protocol Name: Apptm1Dbo Method: Melt Curve Analysis Version: 3.0 Created: 27-May -2009 Updated: 24-September-2009

Notes: Melting curve analysis is done using a Roche Light Cycler 480. Expected Results: Mutant Tm = 89°C Wild type Tm = 92°C

Gel Image

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Protocol Primers

Primer 5' Label Sequence 5' --> 3' 3' Label Primer Type oIMR1636 - AGA GCA CCG GGA GCA GAG - Wild type oIMR1637 - AGC AGG AGC AGT GCC AAG - Wild type oIMR6916 - CTT GGG TGG AGA GGC TAT TC - Mutant Forward oIMR6917 - AGG TGA GAT GAC AGG AGA TC - Mutant Reverse

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 2.00 - 2.00 2 X SYBR Green Master Mix (Roche) 6.00 1.00 X 6.00 20 uM oIMR6916 0.30 0.50 uM 0.30 20 uM oIMR6917 0.30 0.50 uM 0.30 20 uM oIMR1636 0.70 1.17 uM 0.70 20 uM oIMR1637 0.70 1.17 uM 0.70 DNA 2.00 - 2.00

Cycling  Step # Temp °C Time Note 1 94 10 min - 2 94 30 sec - 3 64 1 min - 4 72 45 sec 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 004133 B6.129S7-Apptm1Dbo/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.