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Mutant Tm = 81°C +/- 1.0°C
Wild type Tm = 87°C +/- 1.0°C
Mutant = 196 bp
Heterozygote = 297 bp and 196 bp
Wild type = 297 bp
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
oIMR9020 | AAG GGA GCT GCA GTG GAG TA | Wild type Forward | A | |||
oIMR9021 | CCG AAA ATC TGT GGG AAG TC | Wild type Reverse | A | |||
oIMR9103 | GGC ATT AAA GCA GCG TAT CC | Mutant Reverse | A | WPRE | ||
oIMR9105 | CTG TTC CTG TAC GGC ATG G | Mutant Forward | A | tdTomato |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTP KAPA | 0.26 mM |
oIMR9020 | 0.50 uM |
oIMR9021 | 0.50 uM |
oIMR9103 | 0.50 uM |
oIMR9105 | 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 |
---|---|
031800 | B6.Cg-Gad2tm1Rpa Gad1tm1Rpa Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/RpaJ |
007914 | B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J |
012567 | B6.Cg-Gt(ROSA)26Sortm27.1(CAG-COP4*H134R/tdTomato)Hze/J |
032864 | B6.Cg-Gt(ROSA)26Sortm65.2(CAG-tdTomato)Hze/J |
025106 | B6.Cg-Gt(ROSA)26Sortm75.1(CAG-tdTomato*)Hze/J |
007909 | B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J |
008538 | B6.Cg-Tg(Cspg4-cre/Esr1*)BAkik/J |
008863 | B6.Cg-Tg(Tek-cre)1Ywa/J |
012570 | B6;129S-Gt(ROSA)26Sortm34.1(CAG-Syp/tdTomato)Hze/J |
021875 | B6;129S-Gt(ROSA)26Sortm65.1(CAG-tdTomato)Hze/J |
021876 | B6;129S-Gt(ROSA)26Sortm66.1(CAG-tdTomato)Hze/J |
007908 | B6;129S6-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J |
007905 | B6;129S6-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J |
030855 | STOCK Notch1tm2Rko Notch2tm3Grid Gt(ROSA)26Sortm9(CAG-tdTomato)Hze Tg(KRT5-cre/ERT2)2Ipc/JeswJ |
14 strains use this protocol |