Stock Number: 013786 Strain Name: B6.129S1(Cg)-Lama2tm1Eeng/J

Allele: Lama2tm1Eeng Protocol Name: Lama2tm1Eeng Method: Standard PCR Version: 1.0 Created: 08-November -2010 Updated: 26-January -2011

Notes: This assay does not work well without the use of a Hotstart Taq polymerase. Expected Results: Mutant = 446 bp Heterozygote = 246 bp and 446 bp Wild type = 246 bp

Gel Image

Image Image file View Full Size View Full Size

Protocol Primers

Primer 5' Label Sequence 5' --> 3' 3' Label Primer Type 11357 - ACT GCC CTT TCT CAC CCA CCC TT - Common 11358 - GTT GAT GCG CTT GGG AC - Wild type Reverse 11359 - CGA CAG TAT CGG CCT CAG - Mutant Reverse

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 2.10 - 2.10 2.5 mM dNTP 0.96 0.20 mM 0.96 5 mM DNA Loading Dye 1.66 0.69 mM 1.66 25 mM MgCl2 0.96 2.00 mM 0.96 DNA 2.00 - 2.00 5 X KAPA LR HotStart Buffer 2.40 1.00 X 2.40 20 uM 11357 0.60 1.00 uM 0.60 20 uM 11358 0.60 1.00 uM 0.60 20 uM 11359 0.60 1.00 uM 0.60 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 58 30sec - 4 72 30sec - 5 - - Go to step 2-4, 35 times 6 72 2 min - 7 10 - hold

This genotyping protocol is used for the following strains:   Stock Number Strain Name 013786 B6.129S1(Cg)-Lama2tm1Eeng/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.