Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr15:31606344+31606430 87bp TCATGAGGCTTCTGCAAAGAT GGTGGGACAACAGTTTAGCC Mut= 92 bpWt= 87 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletions in lower case):
ACACACACTCGAACACACACACACACACAGACACACAGACACACACACACACACACTCTCTCTCTCATACACACACACACACACTCTCTCTCTCTCTCTCTCACACACACACACACACACACACACACACACACACACACACACacGCAGCACGAGTCAGTGTGAGAAGGGCTGCTGAGAAGatgcagccttggctttctagtgtgtgctccaagtgtttctgctttcccccaatgatggctcaaatctaggaacgcctgaggaagaaagacaggcgggccctgttcttcctacgagtctggaatccgacagttcaaagaggaccagttggggattcttgattactggggtcgttggtggtgcactgctgacagtgtatgctgtggccacaccattcatcaccccggccctccggaaagtttgtttgccattcgtgcctgcaacttcgaagcaggttgaaaatgttgtcaggatgctgcgacacagaagaggacccctggtggacatcggcagtggcgatgggcggattgtgagttatcctaaaacattttaaggttaaactttaaccttaaaccttaataagagtggccttattcgaggaggtgtatcactgggggcacgctttgaggtttcaaaagcccaagccattcccagttagctgttaaactttctgctttatttttttcatgaggcttctgcaaagatttgccaattgtggcagttggaaaagtgtgtgtcttattttgcgtttggctaaactgttgtcccaccagcATGTGTTCAGAGTCCATTTTTCTGTGATTGATTTTTCCTCCCCTGGAGGAAAAGAACTCTAAACATGCCAGATAGAAAGGAGCAGTTAATTGCTAATTATAAAGTAGTCTATTAGGATTTTTGGTCCAGTAAAGGTGGCATGATCACA
This mutation is a 592 bp deletion beginning at Chromosome 15 position 31605842 bp and ending after 31606433 bp (GRCm38/mm10). In addition there is a 2 bp deletion [GT]36 bp after the 592 deletion.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
45552 | GTG TGA GAA GGG CTG CTG AG | Mutant Forward | A | |||
45553 | GGC ATG TTT AGA GTT CTT TTC CTC | Mutant Reverse | A | |||
45555 | Fluorophore-1 | TGT GAT TGA TTT TTC CTC CCC | Quencher-1 | MUT Probe | ||
46472 | TCA TGA GGC TTC TGC AAA GAT | Wild type Forward | A | |||
46473 | GGT GGG ACA ACA GTT TAG CC | Wild type Reverse | A | |||
46474 | Fluorophore-2 | ATT GTG GCA GTT GGA AAA GTG T | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
45552 | 0.40 uM |
45553 | 0.40 uM |
46472 | 0.40 uM |
46473 | 0.40 uM |
Wt Probe | 0.15 uM |
Mutant Probe | 0.15 uM |
DNA |
Step | Temp °C | Time | Note |
---|---|---|---|
1 | 95.0 | -- | |
2 | 95.0 | -- | |
3 | 60.0 | -- | |
4 | -- | repeat steps 2-3 for 40 cycles | |
5 | 40.0 | -- | Forever |