Stock Number: 007900 Strain Name: C57BL/6-Gt(ROSA)26Sortm1(HBEGF)Awai/J

Allele: Gt(ROSA)26Sortm1(HBEGF)Awai Protocol Name: Gt(ROSA)26Sortm1(HBEGF)Awai Method: Standard PCR Version: 2.0 Created: 09-September-2011 Updated: 09-September-2011

Expected Results: Mutant = 242 bp Heterozygote = 242 bp and 603 bp Wild type = 603 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 oIMR8128 - CAT CAA GGA AAC CCT GGA CTA CTG - Mutant Reverse oIMR8545 - AAA GTC GCT CTG AGT TGT TAT - Common oIMR8546 - GGA GCG GGA GAA ATG GAT ATG - Wild type Reverse

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 2.40 - 2.40 25 mM MgCl2 0.96 2.00 mM 0.96 2.5 mM dNTP 0.96 0.20 mM 0.96 20 uM oIMR8545 0.60 1.00 uM 0.60 20 uM oIMR8546 0.60 1.00 uM 0.60 20 uM oIMR8128 0.30 0.50 uM 0.30 5 mM DNA Loading Dye 1.66 0.69 mM 1.66 DNA 2.00 - 2.00 5 X KAPA LR HotStart Buffer 2.40 1.00 X 2.40 2.5 U/ul KAPA LR HotStart Taq 0.12 0.03 U/ul 0.12

Cycling  Step # Temp °C Time Note 1 94 3 min - 2 94 30 sec - 3 61 1 min - 4 72 1 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 007900 C57BL/6-Gt(ROSA)26Sortm1(HBEGF)Awai/J 008040 CBy.B6-Gt(ROSA)26Sortm1(HBEGF)Awai/J 016603 NOD.B6-Gt(ROSA)26Sortm1(HBEGF)Awai/DvsJ Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.