Stock Number:014539
Strain Name:B6;129S-Gt(ROSA)26Sortm39(CAG-hop/EYFP)Hze/J
CAG-HOP/EYFP)Hze
CAG-HOP/EYFP

Separated MCA

2.0
09-May -2012 09-May -2012
Notes:

This assay does not work well without the use of a Hotstart Taq polymerase.
Expected Results:

Mutant = 190 bp

Heterozygote = 190 bp and 242 bp

Wild type = 242 bp

Melt Peaks
Wt = 88.3°C +/- 1
Mut = 87.7° C+/- 1
Gel Image 
Protocol Primers 
Primer 5' Label Sequence 5' --> 3' 3' Label Primer Type
12177 - CTT TAA GCC TGC CCA GAA GA - Wild type
12180 - ATA TCC TGC TGG TGG AGT GG - Mutant
12181 - GCC ACG ATA TCC AGG AAA GA - Mutant
oIMR8713 - TCC CAA AGT CGC TCT GAG TT - Wild type
Reaction/Components 
Reaction Component Volume (µl) Final
Concentration		 Total Volume (µl)
ddH2O 3.95 - 3.95
5 X Kapa 2G HS buffer 2.40 1.00 X 2.40
25 mM MgCl2 0.96 2.00 mM 0.96
10 mM dNTP KAPA 0.24 0.20 mM 0.24
20 uM oIMR8713 0.30 0.50 uM 0.30
20 uM 12177 0.30 0.50 uM 0.30
5 mM 10x Loading Dye 1.20 0.50 mM 1.20
2.5 ul Kapa 2G HS taq polymerase 0.05 0.01 ul 0.05
20 X EvaGreen 0.60 1.00 X 0.60
DNA 2.00 - 2.00
Cycling 
Step # Temp °C Time Note
1 94 2 min -
2 94 20sec -
3 65 15sec -1.5 C per cycle
4 68 10sec -
5 - - repeat steps 2-4 for 10 cycles
6 94 15sec -
7 50 15sec -
8 72 10sec -
9 - - repeat steps 6-8 for 28 cycles
10 72 2 min -
11 10 - hold
Reaction/Components 
Reaction Component Volume (µl) Final
Concentration		 Total Volume (µl)
ddH2O 3.95 - 3.95
5 X Kapa 2G HS buffer 2.40 1.00 X 2.40
25 mM MgCl2 0.96 2.00 mM 0.96
10 mM dNTP KAPA 0.24 0.20 mM 0.24
20 uM 12180 0.30 0.50 uM 0.30
20 uM 12181 0.30 0.50 uM 0.30
5 mM 10x Loading Dye 1.20 0.50 mM 1.20
2.5 ul Kapa 2G HS taq polymerase 0.05 0.01 ul 0.05
20 X EvaGreen 0.60 1.00 X 0.60
DNA 2.00 - 2.00
This genotyping protocol is used for the following strains:
 
Stock Number Strain Name
014539 B6;129S-Gt(ROSA)26Sortm39(CAG-hop/EYFP)Hze/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 2 min -
2 94 20sec -
3 65 15sec -1.5 C per cycle
4 68 10sec -
5 - - repeat steps 2-4 for 10 cycles
6 94 15sec -
7 50 15sec -
8 72 10sec -
9 - - repeat steps 6-8 for 28 cycles
10 72 2 min -
11 10 - hold