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Mut = A (CBA allele)
WT = G(B6 allele)
GGCATAACCTACAACTGCGGGGCCTGGTC(g/a)TGCTCCTCATATCAAAGGCTGCGGGG
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
pIMR344 | Fluorophore | GCC ATC TAC AGC TTG ATC TGT CC | ||||
pIMR345 | ATG AGT ATA CCC CAC TGA CCA TGT | |||||
pIMR346 | GCC TTT GAT ATG AGG AGC |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.00 |
MgCl2 | 2.00 |
dNTPS-kapa | 0.20 |
pIMR344 | 0.50 |
pIMR345 | 0.50 |
Glycerol | 5.00 |
Kapa 2G HS taq polym | 0.01 |
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 | |
6 | 94.0 | -- | |
7 | 60.0 | -- | |
8 | 72.0 | -- | |
9 | -- | repeat steps 6-8 for 28 cycles | |
10 | 72.0 | -- | |
11 | 10.0 | -- | hold |