Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 88 bp
Wild Type = 148 bp
>chr9:44853132-44853279 148bp TCTTTGAAAGGTTTGGACCTG CAAACAGTCTATAAAGTCACAGAGTTG
Wt Sequence (with deletion in lowercase):
TTATGTTATTATTCCCTATATGTCTGTAGCTATAAGGACTTGCTATATTTGTGGCTGTGTATTTTTATACTGATTTGCTTATTTGTATTAAAATTATCTTTGAAAGGTTTGGACCTGGACCtgtagtttgaaattcaggactattccttatctaatgctcaccagagagaagctttctatgcattgctgagtgatcacacagctactatgtgtgatgcaactctgtgactttatagactgtttggataatgatgattgggatgcacacaccaattttccaatgcatttgtttcatctaggcctttcctttggatttattcacatttatgtttttgtttctgattttaaaataattttcctttgttgtaggatgagcagttcttaggttttggctcagatgaagaagtcagagtgcgaagccccaccaggtctccttcaggtatggccaataaagcacgtgtcacagatctgcattctgtctgttaggagagtatggcttcctcctgccttgttgttagtaaggatgattgcagccCAGGCCAGGAAACTGTGTGAGCTAGACTGGGCAGACTGGTTCCTGCTGCCTGTGCTTGTAAATTCTTCACTCTGTTTTCCTGGGTAATACTTGGGCATGGGAAGTAGGTTTCTACAGAAAGATTTTGGGAA
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51688 | TCT TTG AAA GGT TTG GAC CTG | Common | A | |||
51689 | CAA ACA GTC TAT AAA GTC ACA GAG TTG | Wild type Reverse | A | |||
51690 | Fluorophore-1 | AGC TTT CTA TGC ATT GCT GAG TG | Quencher-1 | WT Probe | ||
51691 | ATT TAC AAG CAC AGG CAG CA | Mutant Reverse | A | |||
51692 | Fluorophore-2 | CCA GGA AAC TGT GTG AGC TAG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51688 | 0.40 uM |
51689 | 0.40 uM |
51691 | 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 |