Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr4:116144083-116144188 106bp CCTGGGTTGAAAGCAGAGTG TCCAAGAGATGTGCCTCCTAC
Mut= 104 bp
Wt= 106 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GCAGCCGTGTTACCTCGATTGGGGCTACTTTTCATCCAGAAAATATTTATTAGCTTCCTGCTGCGTGTCTAGTTGGTGCCAACACAGGGAGAAGGCACTGAGCTTTCTTTGAGGTCTTCAGTTCCAAATCCAGGATCACCTAGTCGAACGCCTCCAGGGCTAGAAGTTGTgtgcataccactcggtacctcgctcccccaactgcgcggcttttctgagcgttacggcgccctcctcctgttcccccaccccaccccccgcgcggggcgggcggggatccacgggtttcgctccgcgttcgtagcagagaagggaggacccttgggaaccggaccctggtcgcccagtcctgttctggcttgggcccacatggaggggaccgcggagtcccagacgccggacttgcgagacgtggagggcaaggtgggcaggaagatccctgaaggactgctccgcgggcttcgaggcgagtgcgagctgggaacctctggggacgtgctgctcccgggggcgcccagcaccggccacggcctaggggacaagatcatggcgctgaggatggagctggtgagtgtgggtgctacaggccacggggaacgggatatttgtaggggcaggggcaggttcccggagttgggctggagaagagggtccttccaagatcaatccatacatcctgggttgaaagcagagtgaagtgacgtctcattatttgtctcccaagggtgaatgaaggGTTGATAGGTCCTGAGAACTGAGAGTAGGAGGCACATCTCTTGGAAGTTTTCCCTGCCCAATAATCGGAAGGAGAGAGGCGAGGGCTAAGTAGCACACAAAAAAAACTTAGTACAGCGCAAATTCTTCCCATCGGCATCCGGGGCCACCAATTTCTTCAGACTCCACTATAAGGCAACCG
This mutation is a 566 bp deletion beginning at Chromosome 4 position 116,144,128 bp and ending after 116,144,693 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
45721 | CCT GGG TTG AAA GCA GAG TG | Wild type Forward | A | |||
45722 | TCC AAG AGA TGT GCC TCC TAC | Common | A | |||
45723 | AGG TCT TCA GTT CCA AAT CCA | Mutant Forward | A | |||
45724 | Fluorophore-1 | TCT CCC AAG GGT GAA TGA AG | Quencher-1 | WT Probe | ||
45725 | Fluorophore-2 | AGG GCT AGA AGT TGT GTT GAT AGG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
45721 | 0.40 uM |
45722 | 0.40 uM |
45723 | 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 |