Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Wt Sequence (deletions in lower case):
CACTAGTCTCGTGCCCAGCATCTCTCCTCCCCACGCACCGACAACCCTGGGCCGCcgcggggcgggggatgccgggctgccgaatcagcgcctgcggcccaggggcccaggaagggacggcagaaccggggtcgcccccgccgccaccccgggagcccctgccgtccctccagcccccttccccatcgccgacctcgactccgacccccactaagtcaccgcccttacccgaggcggccgagacacccgtggaagggcaggagctgcagcgctggcgccagggtgctagtggggggtctgggggcgcgggcccggcggggatcgcgggcgcggcggcgggggcagggggccgtgcgctggagctggccgaagcgcggcggcgactgctagaagtggagggccgccggcgcctggtgtcggagctggagagccgggtgctgcagctgcaccgagtcttcttggctgcggagctgcgcctggcgcatcgcgccgagagcctgagccgcctgagcggcggcgtggcccaggccgagctctacctggctgcgcacgggtcccgtctcaagaagggtgcgcgccgcggtcgccggggtcgtcccccggcgctgctggcgtccgcgctcggcctggggagctgcgtgccctggggcgcagggcggctacggcgcggccagggcccggagcccgactcgcccttccgccgcAGCCCGCCCCGCGGCCCCGCCTCCCCCCAGCGCTGACCTCAGTACCCCGGACCCCTGGCTTCCCGAGGCAGGCGAGTCTCCAGGTGCGGGAGGAC
This mutation is a 649 bp deletion beginning at Chromosome 4 position 133,497,822 bp and ending after 133,498,470 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51541 | AGT CTC GTG CCC AGC ATC TC | Common | A, B | |||
51542 | TTC TGC CGT CCC TTC CTG | Wild type Reverse | A | |||
51543 | CTC CCG CAC CTG GAG ACT | Mutant Reverse | B |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTPS-kapa | 0.26 mM |
51541 | 0.50 uM |
51542 | 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 |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTPS-kapa | 0.26 mM |
51541 | 0.50 uM |
51543 | 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 |