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Mutant = ~400 bp
Heterozygote = 377 bp and ~400 bp
Wild type = 377 bp
Wt Sequence: tagatagcctctcacactgtagccaaggctggtctaGatctcactatgtaggcccaactag
Mutant Sequence: catgctccagactgccttgggaaaagcgcctcccctacccggtagaattcataacttcgtatagcatacattatacgaagttatggatcccccGATCTCACTATGTAGGCCCAACTAGC
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
12834 | GCT ACT TCC ATT TGT CAC GTC C | Mutant Forward | ||||
15294 | TCT GGG ACA GCA AGG TAT GA | Wild type Forward | ||||
15295 | GGC CAC TAA CAT TTC CCT TTC | Common |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.00 |
MgCl2 | 2.00 |
dNTPS-kapa | 0.20 |
Primer 1 | 0.50 |
Primer 2 | 0.50 |
10x Loading Dye | 0.50 |
Kapa 2G HS taq polym | 0.01 |
DNA |
Step | Temp °C | Time | Note |
---|---|---|---|
1 | 94.0 | -- | |
2 | 94.0 | -- | |
3 | 65.0 | -- | -1.5 C per cycle decrease |
4 | 68.0 | -- | |
5 | -- | Go to step 2-4, 10 times | |
6 | 94.0 | -- | |
7 | 50.0 | -- | |
8 | 72.0 | -- | |
9 | -- | Go to step 6-8, 28 times | |
10 | 72.0 | -- | |
11 | 10.0 | -- | hold |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.00 |
MgCl2 | 2.00 |
dNTPS-kapa | 0.20 |
Primer 3 | 0.50 |
Primer 4 | 0.50 |
10x Loading Dye | 0.50 |
Kapa 2G HS taq polym | 0.01 |
DNA |
Step | Temp °C | Time | Note |
---|---|---|---|
1 | 94.0 | -- | |
2 | 94.0 | -- | |
3 | 65.0 | -- | -1.5 C per cycle decrease |
4 | 68.0 | -- | |
5 | -- | Go to Step 2-4, 10 times | |
6 | 94.0 | -- | |
7 | 50.0 | -- | |
8 | 72.0 | -- | |
9 | -- | Go to Step 6-8, 28 times | |
10 | 72.0 | -- | |
11 | 10.0 | -- | hold |