Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr19:47075162+47075306 145bp AGGAGCAAACTGACAACAATG CCCAAATCCTCACATATACCA
Mut= 125 bp
Wt= 145 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
AGTGTTGGGATTAAAGAAAGACGTGCACCACCATGGGTCAAAGATGGTTTTCAAAAGAAATACATACTGGTAGTGAGTCTTAGGTCTGCTCAGAAGCCCCATTCACTGCATAATTCCAGAAGGTTCTCCTGAAGTGCTTATCATACGTTTCAGTAGAGAAATGGGTGGCTTTAGAAACTGAGTTATAATCATTTTGAAATATATTACTATTGATCCTACAgcctagtcagggttatttatatagttccattattacaaatatataattatatgctagctaccgatgattctgggatggtttataaatgtatgagggagaagctgattaccttaatgacacattgaaaaatgactctttaaaaatcactaaaacaggttccatggagatcaggaatgttactaccaggatgacctacgagtgttatctagtcttaccaaaaaggagcacatgaaagggaacgagaccatgctggactttcggacgagtaaattcgttctccgcatttcccgggactcgtaccagctcttgaagcggcatctccaggagaagcagaacaatcagatctggaacatcgtgcaggagcacctctacatcgacatcttcgacgggatgcctcgcagtaagcaacagatcgacgccatggtgggcagcctggcaggagaggcgaaacgagaggcgaacaaatcgaaggtacgggagctaacctagaatgatgtaatgtgcaggagcaaactgacaacaatgtcatgtgagtttccattatataatggaaattatgtatacattcttataactaccttggGAGGAATCCAGTACTGCATTTAAAATGTGTTGTTCTGAAGATTTGTGGTATATGTGAGGATTTGGGAATGGTTTCGAGTAACAGAGCCTTAACTGCAGAGGCTAAGACGAGCAAGGTTTGTTTGTTTTTTTTTTTTTTTTTTTTGCTTTTGCTTTTGCTTCTGAGACCCGCATAATAATTCCAAAGTGCAGTCTATACCTCTTGGCTTGTGGCTCCT
This mutation is a 583 bp deletion beginning at Chromosome 19 position 47,074,658 bp and ending after 47,075,240 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48689 | AGG AGC AAA CTG ACA ACA ATG | Wild type Forward | A | |||
48690 | CCC AAA TCC TCA CAT ATA CCA | Common | A | |||
48691 | TGG GTG GCT TTA GAA ACT GA | Mutant Forward | A | |||
48692 | Fluorophore-1 | CAT GTG AGT TTC CAT TAT ATA ATG GAA A | Quencher-1 | WT Probe | ||
48693 | Fluorophore-2 | CTA TTG ATC CTA CAG AGG AAT CCA GT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48689 | 0.40 uM |
48690 | 0.40 uM |
48691 | 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 |