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Mutant = 330 bp
Heterozygote = 330 bp and 468 bp
Wild type = 468 bp
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
19693 | TCC CCT GGC ACA ACG TAA G | Mutant Reverse | A | chicken HS4 chromatin insulator | ||
22159 | GTG TAG CCC TGG CTT TTC TG | Common | A | |||
22330 | GAA CTC ACA GTG GCC AGT CA | Wild type Reverse | A |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTP KAPA | 0.26 mM |
19693 | 0.50 uM |
22159 | 0.50 uM |
22330 | 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 |
---|---|
024115 | B6.Cg-Igs7tm94.1(tetO-GCaMP6s)Hze Tg(Camk2a-tTA)1Mmay/J |
024104 | B6.Cg-Igs7tm94.1(tetO-GCaMP6s)Hze/J |
024103 | B6;129S6-Igs7tm93.1(tetO-GCaMP6f)Hze/J |
024107 | STOCK Gt(ROSA)26Sortm5(ACTB-tTA)Luo Igs7tm93.1(tetO-GCaMP6f)Hze/HzeJ |
024112 | STOCK Gt(ROSA)26Sortm5(ACTB-tTA)Luo Igs7tm94.1(tetO-GCaMP6s)Hze/J |
5 strains use this protocol |