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Mutant = ~110 bp
Heterozygote = ~110 bp and 241 bp
Wild type = 241 bp
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
17772 | TGC CGG TCC TAT TTA CTC GT | Mutant Forward | A | Flp | ||
17773 | TAC TTC TTT AGC GCA AGG GGT AG | Mutant Reverse | A | Flp | ||
24500 | CAG GAC AAC GCC CAC ACA | Wild type Reverse | A | |||
oIMR9020 | AAG GGA GCT GCA GTG GAG TA | Wild type Forward | A |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTP KAPA | 0.26 mM |
17772 | 0.50 uM |
17773 | 0.50 uM |
24500 | 0.50 uM |
oIMR9020 | 0.50 uM |
Glycerol | 6.50 % |
Dye | 1.00 X |
Kapa 2G HS taq polym | 0.03 U/ul |
DNA |
Step | Temp °C | Time | Note |
---|---|---|---|
1 | 94.0 | -- | |
2 | 94.0 | -- | |
3 | 65.0 | -- | -0.5 C per cycle decrease |
4 | 68.0 | -- | |
5 | -- | repeat steps 2-4 for 10 cycles (Touchdown) | |
6 | 94.0 | -- | |
7 | 60.0 | -- | |
8 | 72.0 | -- | |
9 | -- | repeat steps 6-8 for 28 cycles | |
10 | 72.0 | -- | |
11 | 10.0 | -- | hold |
Stock Number | Strain Name |
---|---|
003946 | 129S4/SvJaeSor-Gt(ROSA)26Sortm1(FLP1)Dym/J |
009086 | B6.129S4-Gt(ROSA)26Sortm1(FLP1)Dym/RainJ |
032468 | B6;129S-Trhtm1.1(cre)Mjkr/LowlJ |
030934 | B6N(129S4)-Sumf1tm1c(KOMP)Wtsi/LutzyJ |
016226 | B6N.129S4-Gt(ROSA)26Sortm1(FLP1)Dym/J |
5 strains use this protocol |