Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr11:22172724-22172872 149bp TCTCCAGTTCTCACTGTCCTG GCATTCATCCTCTGAAAACA
Mut= 147 bp
Wt= 149 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletions in lower case):
TTATAGGTTTAGCAAGAAAACACCATTTGATTACATTAATTTAATCAAAGAGAGAAAAAAAGCAGGTAAGGGCACTTACAGTGCCCTTCTTGCCTATGATAGATCTTTTTCTCACACTAGTCTTGGAGCCATTCCCAGTTTTCCTGAGGCAGGGTCTTACATAGTTGAGGCTAGCTGTGAATTCTGGATCCTTGTGCCTGTATCTACCTTAGTGCTGGACTTATAGGTGTGAGCTCTACGTGTGCTCGTCACTAGTTTCTGTTTCACATTCTCCTCTagtataaatatttatgcctgggccatctctttctgttttacttagatataaaagctgtaataaaagctcatcacaattttattattgcattacaagaatttttttagcaaaggaagttcttgtttcttttttattggctataaaataattccaaactacataattatttttttgtcttccaggaatccccttctggtcggaggaaagctcttgccaccagcagcattaacatgaaacagtacgcaagccccatgccaacacaaactgacgtcaagttaaaattcaagccgctgtctaagaaggtcgtgtctgccactctccagttctcactgtcctgtatcttccttcgggagggaaaagcaacgtaagtgactcccttCCCGTcttagtGGATGGGCGCAGGGCAGACATCCATGCACGCCAGCCCTTCTCCAGCTCTCTGCTTTGTTTTCAGAGGATGAATGCCCTTGTTATTTTAAAATCCTGTCTTGATTCTCCAGAGCAACAATGACATAAAAAGTGTAACTGTTACCATTTCAAAATCAGTATGAGTTGACCTAGTGATGTGCACCCCAGTCCCAGCACTCAAGGCAAAAACATTTGGTACCACTGGCTGCCATTTTTGTC
This mutation is a 379 bp deletion beginning at Chromosome 11 position 22,172,810 bp and ending after 22,173,188 bp (GRCm38/mm10). In addition, there is a 6 bp deletion CTTAGT 4 bp after the 379 bp deletion.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48979 | GCA TTC ATC CTC TGA AAA CA | Common | A | |||
50477 | TCT CCA GTT CTC ACT GTC CTG | Wild type Forward | A | |||
50478 | AGT GCT GGA CTT ATA GGT GTG AG | Mutant Forward | A | |||
50479 | Fluorophore-1 | CCT TCG GGA GGG AAA AGC | Quencher-1 | WT Probe | ||
50480 | Fluorophore-2 | ATT CTC CTC TCC CGT GGA TGG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48979 | 0.40 uM |
50477 | 0.40 uM |
50478 | 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 |