Stock Number: 006942 Strain Name: B6.129-Cd33tm1Ajv/J

Allele: Cd33tm1Ajv Protocol Name: Cd33tm1Ajv Method: Separated PCR Version: 1.1 Created: 03-July -2008 Updated: 24-September-2009

Expected Results: Mutant = 500 bp Heterozygote = 500 bp and 1048 bp Wild type = 1048 bp

Gel Image

Image Image file View Full Size

Gel Information: Separated by gel electrophoresis on a 1.5% agarose gel.

Protocol Primers

Primer 5' Label Sequence 5' --> 3' 3' Label Primer Type oIMR8001 - GGT CCA CAC CAG GGA CAA CTG - Common oIMR8002 - CGG GCG CCA CCA GCT GGA ATT C - Wild type Reverse oIMR8003 - GGA GGA GTA GAA GGT GGC G - Mutant Reverse

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 3.36 - 3.36 10 X PCR Buffer II (ABI) 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 oIMR8001 0.90 1.50 uM 0.90 20 uM oIMR8003 0.90 1.50 uM 0.90 5 M Betaine 1.66 0.69 M 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 64 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 3.76 - 3.76 10 X PCR Buffer II (ABI) 0.80 0.67 X 0.80 25 mM MgCl2 0.96 2.00 mM 0.96 2.5 mM dNTP 0.96 0.20 mM 0.96 20 uM oIMR8001 0.90 1.50 uM 0.90 20 uM oIMR8002 0.90 1.50 uM 0.90 5 M Betaine 1.66 0.69 M 1.66 5 U/ul Taq DNA Polymerase 0.06 0.03 U/ul 0.06 DNA 2.00 - 2.00 This genotyping protocol is used for the following strains:   Stock Number Strain Name 006942 B6.129-Cd33tm1Ajv/J 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 64 1 min - 4 72 1 min repeat steps 2-4 for 35 cycles 5 72 2 min - 6 10 - hold**