Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:125666304-125666423 120bp TGTCTAAGGCTTTGGCTCAGT GGCAACATAGTCTGTGGTATCCTAT
Mutant= 105 bp
Wild Type = 120 bp
Wt Sequence (deletions in lower case):
CAACATTAACGGGCTGAGAACTTATGCCATAGAAAAACAGTTATGTCTAAGGCTTTGGCTCAGTTGGGTCctaaggaagtcttatcttattcattcattttaaatgcaattgtttctctctctccctctctttcttttataggataccacagactatgttgcctatgtagctaaagatccagtcaaccaacgaggtatggacaagaaacttctgggttttcataagagctgttagctaccgactataaaaataagtggttatttctaatgtacacagtaatgggtgtcacacagtaaggccctatctatgggcattgctttctgttatcttaggagtcatggtcattgcagacttgagcactcttgctaagctgaacaggcgataaagagaagctgtgaggcccctcctggcctgtttcatagtcttccaaacgatgggcatggtccaatggtgccccatcattgtaggatgatattgaaggactctaagaaaactatgtaaattttaattgaaaataaataatcctaccagcagagaagagtggaacaattaacaggcaatatttaaattccagcacttgaaagattcACAAACTAAAAAGCATATTTACATATGCAAAAATAAGCTGTTTTGTTTATGGTAGTGTTGTTCCCATATCCCAAGGAGAAAAAGAAATTGAACACAAAACTCAATGAGACATTGCTGTTGGTGTGTGTGTGTGTGTG
This is a 519 bp deletion beginning at Chromosome 2 position 125,665,878 bp and ending after 125,666,396 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44276 | CTC CTT GGG ATA TGG GAA CA | Mutant Reverse | A | |||
46744 | Fluorophore-1 | TGG GTC ACA AAC TAA AAA GCA TAT T | Quencher-1 | MUT Probe | ||
46745 | Fluorophore-2 | TCT TAT TCA TTC ATT TTA AAT GCA ATT G | Quencher-2 | WT Probe | ||
46746 | TGT CTA AGG CTT TGG CTC AGT | Common | A | |||
46747 | GGC AAC ATA GTC TGT GGT ATC CTA T | Wild type Reverse | A |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44276 | 0.40 uM |
46746 | 0.40 uM |
46747 | 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 |